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Gene regulatory networks. Interactive diagrams for the web-browser-based exploration of LCH-subset-specific gene regulatory networks.

Supplementary Figures

Marker genes and genomic regions. Lists of differentially expressed genes between LCH cells and non-LCH immune cells, marker genes for each LCH cell subset, and differentially accessible genomic regions between LCH cell subsets.

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions.Significance:This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy.See related commentary by Gruber et al., p. 1343.This article is highlighted in the In This Issue feature, p. 1325

Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a+langerin+ cells of unknown origin. We have previously reported that the Notch signaling pathway is active in LCH lesions and that the Notch ligand Jagged2 (JAG2) induces CD1a and langerin expression in monocytes in vitro. Here we show that Notch signaling induces monocytes to acquire an LCH gene signature and that Notch inhibition suppresses the LCH phenotype. In contrast, while also CD1c+ dendritic cells or IL-4-stimulated CD14+ monocytes acquire CD1a and langerin positivity in culture, their gene expression profiles and surface phenotypes are more different from primary LCH cells. We propose a model where CD14+ monocytes serve as LCH cell precursor and JAG2-mediated activation of the Notch signaling pathway initiates a differentiation of monocytes toward LCH cells in selected niches and thereby contributes to LCH pathogenesis. This study aims to identify the cell of origin of Langerhans Cell Histiocytosis and the molecular mechanisms of its pathogenesis.

Langerhans cell histiocytosis (LCH) is a rare neoplasm predominantly affecting children. It occupies a hybrid position between cancers and inflammatory diseases, which makes it an attractive model for studying cancer development. To explore the molecular mechanisms underlying the pathophysiology of LCH and its characteristic clinical heterogeneity, we investigated the transcriptomic and epigenomic diversity in primary LCH lesions. Using single-cell RNA sequencing, we identified multiple recurrent types of LCH cells within these biopsies, including putative LCH progenitor cells and several subsets of differentiated LCH cells. We confirmed the presence of proliferative LCH cells in all analyzed biopsies using IHC, and we defined an epigenomic and gene-regulatory basis of the different LCH-cell subsets by chromatin-accessibility profiling. In summary, our single-cell analysis of LCH uncovered an unexpected degree of cellular, transcriptomic, and epigenomic heterogeneity among LCH cells, indicative of complex developmental hierarchies in LCH lesions. Significance: This study sketches a molecular portrait of LCH lesions by combining single-cell transcriptomics with epigenome profiling. We uncovered extensive cellular heterogeneity, explained in part by an intrinsic developmental hierarchy of LCH cells. Our findings provide new insights and hypotheses for advancing LCH research and a starting point for personalizing therapy. See related commentary by Gruber et al., p. 1343. This article is highlighted in the In This Issue feature, p. 1325

Langerhans cell histiocytosis (LCH) is a histiocytic disorder driven by a constitutive activation of the MAPK signaling pathway in myeloid cells. In 50–60% of cases, it is caused by the BRAFV600E mutation. There is evidence that levels of BRAFV600E in the peripheral blood of patients with LCH correlate with disease burden and could be used as marker for disease extent and response to therapy. However, there is currently no consensus on how testing for minimal disseminated disease should be performed. Different approaches to determine the mutation load in patients with LCH were assessed and longitudinal evaluation of patient DNA during treatment with chemotherapy and/or the RAF inhibitor vemurafenib was performed. DNA was isolated from whole blood, different leukocyte subsets, and circulating cell-free DNA (ccf-DNA). We show that determining BRAF levels from whole blood is superior to using ccfDNA. Furthermore, it is important to identify the clinically relevant BRAF-mutated cellular subpopulations such as CD14+ monocytes or CD1c+ DCs, since other blood cells can also harbor the mutation and therefore confound whole blood or ccfDNA measurements. Our data support the view that single-agent treatment with an RAF inhibitor reduces disease activity but does not cure LCH.

Langerhans cell histiocytosis (LCH) is a MAPK pathway-driven disease characterized by the accumulation of CD1a

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.

The p53 gene is compromised in most human cancers by point mutation. Evidence is accumulating that these alterations frequently do not result in a complete loss of the sequence-specific transcriptional regulatory function of p53. Here, we describe the transcriptional activity of the p53 mutant C277Y isolated from a Ewing's sarcoma with high constitutive pig3 expression. Transient transfection of this mutant into a p53 null cell line resulted in activation not only of the pig3 but also of the MDM2 gene compatible with the presence of constitutively expressed MDM2 transcripts initiated from the P2 promoter in the p53-C277Y hemizygous Ewing's sarcoma cell line. Expression of endogenous pig3 and MDM2 genes was further enhanced on irradiation of this cell line. Here, suppression of p53-C277Y by RNAi reduced pig3 promoter activity, RNA, and protein expression. Reporter gene assays revealed that the potential of p53-C277Y to up-regulate MDM2 expression was similar to wild-type p53, whereas activation of the pig3 promoter was at least 5-fold increased over wild-type p53. The pentanucleotide microsatellite sequence present in exon 1 of the pig3 gene was found to be responsible for p53-C277Y-mediated activation. In concordance with a role of PIG3 protein for cell death, we showed residual apoptotic activity of p53-C277Y to which the described Ewing's sarcoma cell line was found to be resistant. p53-C277Y has previously been reported to bind to DNA with altered sequence specificity and to be unable to activate generic p53 target genes in yeast-based functional assays. Our results, therefore, show that a p53 mutant may behave differently when tested in its authentic cellular context.

The EWS-FLI1 transcription factor is consistently expressed in 85% of Ewing tumors (EFT). In heterologous cells, EWS-FLI1 induces p53-dependent cell cycle arrest or apoptosis. It has been speculated that the p53 tumor suppressor pathway may be generally compromised in EFT despite only rare p53 mutations. In order to test for functional integrity of this pathway, we have investigated a series of EFT cell lines that differ from each other with respect to their endogenous p53 and INK4A gene status for their response to ectopic p53 expression and to stimulation of endogenous p53 activity by X-ray treatment. Significant interindividual and intratumoral variations in the apoptotic propensity of EFT cell lines to transient expression of ectopic p53 were observed, which was independent of the level of p53 expression. In cell lines with a low apoptotic incidence, apoptosis was delayed and the surviving fraction showed a prolonged growth arrest. Complete resistance to p53-induced apoptosis in two cell lines established from the same patient was associated with a high BCL2/BAX ratio and low levels of APAF1. Sensitivity to X-rays showed a trend towards a higher apoptotic rate in wild-type (wt) p53 expressing than in p53 mutant cells. However, one wt p53-expressing EFT cell line was completely refractory to irradiation-stimulated cell death despite high apoptotic responsiveness to ectopic p53. No difference in Ser15 phosphorylation and the transcriptional activation of p53 targets was observed in wt p53 EFT cell lines irrespective of the induction of cell death or growth arrest. All together, our results demonstrate that despite significant variability in the outcome, cell death or cell cycle arrest, the p53 downstream pathway and the DNA damage signaling pathway are functionally intact in EFT.

The pRB cell cycle regulatory cascade is frequently perturbed in neoplasia by overexpression of a component of the pRB-phosphorylating cyclin D1/CDK4 complex or by inactivation of pRB or the CDK4 inhibitors p16 and p15. We investigated the status and expression of p16, p15, CCND1, CDK4 and RB genes in the Ewing family of tumors. P16 loss was observed in 8 of 27 tumors (30%) and in 12 of 23 (52%) tumor cell lines from unrelated patients. There were no discrepancies in the p16 status between primary tumors and the corresponding cell lines and between cell lines established from consecutive tumor samples. p15 was codeleted in most cases but p15 mRNA was absent also in cell lines retaining the gene. In addition, posttranscriptional p16 inactivation was observed in two cases. Although no evidence for CDK4 or CCND1 amplification was obtained, expression of these genes varied considerably in the cell lines in a case specific manner. In wild-type p16 cell lines, pRB expression was lost in one case. Our data indicate that, despite the absence of cytogenetically detectable 9p21 chromosomal aberrations, p16 deletions constitute the most frequent secondary molecular aberration in Ewing tumors so far. These results are discussed in the context of the stage of disease and the clinical outcome of the patients. The potential prognostic impact of these findings remains to be further evaluated.

Deregulated E2F transcription factor activity occurs in the vast majority of human tumors and has been solidly implicated in disturbances of cell cycle control, proliferation, and apoptosis. Aberrant E2F regulatory activity is often caused by impairment of control through pRB function, but little is known about the interplay of other oncoproteins with E2F. Here we show that ETS transcription factor fusions resulting from disease driving rearrangements in Ewing sarcoma (ES) and prostate cancer (PC) are one such class of oncoproteins. We performed an integrative study of genome-wide DNA-binding and transcription data in EWSR1/FLI1 expressing ES and TMPRSS2/ERG containing PC cells. Supported by promoter activity and mutation analyses, we demonstrate that a large fraction of E2F3 target genes are synergistically coregulated by these aberrant ETS proteins. We propose that the oncogenic effect of ETS fusion oncoproteins is in part mediated by the disruptive effect of the E2F–ETS interaction on cell cycle control. Additionally, a detailed analysis of the regulatory targets of the characteristic EWSR1/FLI1 fusion in ES identifies two functionally distinct gene sets. While synergistic regulation in concert with E2F in the promoter of target genes has a generally activating effect, EWSR1/FLI1 binding independent of E2F3 is predominantly associated with repressed differentiation genes. Thus, EWSR1/FLI1 appears to promote oncogenesis by simultaneously promoting cell proliferation and perturbing differentiation.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

The oncogene EWS-FLI1 encodes a chimeric transcription factor expressed in Ewing's sarcoma family tumors (ESFTs). EWS-FLI1 target gene expression is thought to drive ESFT pathogenesis and, therefore, inhibition of EWS-FLI1 activity holds high therapeutic promise. As the activity of many transcription factors is regulated by post-translational modifications, we studied the presence of modifications on EWS-FLI1. The immuno-purified fusion-protein was recognized by an antibody specific for O-linked beta-N-acetylglucosaminylation, and bound readily to a phosphoprotein-specific dye. Inhibition of Ser/Thr-specific phophatases increased EWS-FLI1 molecular weight and reduced its O-GlcNAc content, suggesting that phosphorylation and O-GlcNAcylation of EWS-FLI1 interact dynamically. By mutation analysis, O-GlcNAcylation was delineated to Ser/Thr residues of the amino-terminal EWS transcriptional-activation domain. Metabolic inhibition of the hexosamine biosynthetic pathway abrogated O-GlcNAcylation of EWS-FLI1 and interfered specifically with transcriptional activation of the EWS-FLI1 target Id2. These results suggest that drugs modulating glycosylation of EWS-FLI1 interfere functionally with its activity and might, therefore, constitute promising additions to the current ESFT chemotherapy.

A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21WAF1/CIP1. These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53. [Cancer Res 2008;68(17):7100–9]

mdm2 encodes for an E3 ubiquitin ligase targeting constitutively expressed p53 for proteasomal degradation. Several protein isoforms have been described for human MDM2 (HDM2), some of which may correspond to splicing variants detectable by RT-PCR in many tumors. Upon cellular stress, p53 becomes resistant to MDM2 and, in a feedback loop, up-regulates mdm2 transcription. The physiological relevance of stress-induced mdm2 gene activity is not well understood. We describe a small nuclear RNA of 365 bases comprised of the first five hdm2 exons and lacking polyadenylation. hdm365 precedes full-length hdm2 RNA expression after induction by p53 and accumulates to significant levels in the nucleus, detectable at the site of hdm2 transcription and processing only. Considering a 10-fold lower stability and high steady-state levels of the novel RNA species, hdm365 appears to be the major processing product of hdm2 transcripts. hdm365 induction was observed after ectopic expression of p53 and after DNA damaging treatment of tumor cell lines, primary fibroblasts and lymphocytes, and was not related to apoptosis. Corresponding truncated transcripts were observed in hdm2 amplified cells. High stress-inducible expression levels, absence of a corresponding protein, and nuclear localisation of hdm365 suggest a novel RNA-based function for hdm2.

To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107

p53 triple mutants (120N/121G/277H, 120H/121G/ 277H, 120S/121G/277H and 120H/121G/277Y) have altered sequence specificity in bandshift assays in vitro and transcription assays in vivo. These mutants activate transcription from the site TTT CATG AAA but not from wild type sites. The triple mutants activate more strongly than p53 with a single 277Y mutation. The TTT site matches the wild type p53 consensus at only 4/10 positions and is not recognised by wild type p53. 277Y mutations have been described in human tumours, and Ewing tumour cells expressing this mutant from the endogenous p53 locus selectively activate transcription from transfected luciferase reporters regulated by TTT-mutant p53 binding sites. p53 mutants with altered sequence specificity have potential advantages for cancer gene therapy: if used to activate transcription of conditionally toxic genes they would allow tumour-targeting by p53, which acts as a sensor for the malignant state, but place control over cell killing in the hands of the clinician. Rare tumours expressing such mutants from the endogenous p53 locus could be targeted directly with p53-regulated suicide vectors, but for most tumours both the p53 mutant and the reporter would need to be encoded by the virus.

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of eponymous Langerhans-cell-like cells of unknown origin. It is a rare disease that may affect any age group, although its most severe clinical course affects predominantly young children. The clinical manifestations range from single lesions that can resolve spontaneously to systemic disease that requires intensive chemotherapy and bone marrow transplantation. Here we demonstrate that LCH cells exhibit a unique transcription profile that clearly separates them from indigenous dendritic cells (DCs), i.e. Langerhans cells, myeloid dendritic cells, and plasmacytoid dendritic cells, indicating a distinct DC entity that occurs under pathologic conditions. Molecular analysis revealed that LCH cells selectively expressed NOTCH ligand Jagged 2 (JAG2) and were the only DCs that coexpressed both NOTCH ligand and receptor. In line, activated NOTCH was detected in LCH but not control biopsies. Furthermore, we show that JAG2 signalling induces key LCH markers CD1a and Langerin on MoDCs, suggesting a role of NOTCH signalling in LCH ontogenesis. Interestingly, JAG2 also strongly induced matrix-metalloproteinase (MMP) 1 in MoDCs, which is highly expressed in LCH lesions and mediates massive tissue destruction and remodelling. The MMP induction by JAG2 was selective for DCs and not recapitulated in monocytes. Together these findings strongly suggest that Jagged2 mediated NOTCH activation confers phenotypic and functional aspects of LCH to DCs. Thus, interference with NOTCH signalling may prove an attractive target to combat this disease. 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 2478. doi:1538-7445.AM2012-2478

The EWS-FLI1 chimeric protein, characterizing Ewing's sarcoma (ESFT), is prototypic for an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of recognition motifs for forkhead box (FOX) proteins in EWS-FLI1 repressed promoters. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibiting FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, FOXO1 is excluded from the nucleus as a consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, augmented by chemical inhibition of PI3K, or by mutation of CDK2 or AKT phosphorylation sites. Furthermore, ChIP experiments revealed that EWS-FLI1 directly binds the promoter of FOXO1 in vitro suggesting a multi-layered regulation of FOXO1 expression by transcriptional repression and post-translational modification. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability. A significant overlap between EWS-FLI1 repressed and FOXO1 activated genes was observed. Treatment of ESFT cell lines with Methyl-Seleninic-Acid (MSA) led to re-activation of endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner, and induced massive cell death, which we found to be at least partially FOXO1-dependent. Taken together, these data confirm our hypothesis that a repressive sub-signature of EWS-FLI1 regulated genes is due to suppression of FOXO1. FOXO1 re-activation by small molecules may constitute a novel therapeutic strategy in the treatment of ESFT. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. 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 2961. doi:1538-7445.AM2012-2961

Besides its important role in organismal development, NOTCH receptor signaling exerts tissue specific proliferative or antiproliferative functions. While the oncogenic role of NOTCH has been extensively investigated due to its constitutive activation in T-cell leukemias and several epithelial cancers, little is known about NOTCH mediated tumor suppression. We have previously reported that in Ewing's sarcoma, a pediatric bone tumor driven by the chimeric ETS oncogene EWS-FLI1, auto-stimulatory NOTCH signaling is suppressed and that reactivation results in p53 and consequently growth inhibitory p21 induction via activation of the NOTCH effector HEY1. We now demonstrate that HEY1-mediated p53 stimulation is accompanied by C-terminal p53 acetylation as a consequence of downregulation of nuclear deacetylase sirtuin 1 (SIRT1). We found that both EWS-FLI1 and HEY1 bind to the SIRT1 promoter with opposite transcriptional consequences. Thus, knockdown of EWS-FLI1 and ectopic HEY1 expression resulted in similar SIRT1 modulation and p53 acetylation which could be reversed by ectopically expressed SIRT1. Consistent with these results, treatment of Ewing's sarcoma cell lines with the sirtuin inhibitor Tenovin 6 resulted in massive cell death. Immunohistochemical analysis of more than 310 Ewing's sarcoma samples identified moderate (25%-50% positive nuclei) to strong (>50% positive nuclei) SIRT1 expression in 35% of cases. Additional 20% showed sporadic positivity (between 10-25% positive nuclei). The involvement of SIRT1 in tumor suppressive NOTCH signaling is not restricted to Ewing's sarcoma, but also relevant at least to B-cell malignancies and some normal tissues, since we found that in several B-cell leukemia and lymphoma cell lines and in keratinocytes HEY1 was able to lead to activating p53 acetylation in a SIRT1 suppression-dependent manner. Supported by grants from the Austrian Science Fund (P22328-B09) and the European Comission (EU-FP7 STREP 259348). 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 1190. doi:1538-7445.AM2012-1190

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly binds to 50% of E2F target genes in ESFT. Furthermore, several E2F factors were themselves found to be directly regulated by EWS-FLI1. Therefore, we studied the functional interaction of EWS-FLI1 with E2F. Ten arbitrarily chosen EWS-FLI1/E2F candidate targets including E2F3, RAD51, and ATAD2 were chosen for an in-depth promoter activity study by luciferase reporter gene assays in combination with mutation analysis of transcription factor binding motifs. By ChIP-PCR we confirmed direct EWS-FLI1 promoter binding, and observed decreased reporter activity for all ten studied promoters upon knockdown of EWS-FLI1 by RNAi. Furthermore, the study of promoter occupancy by different E2F family members revealed that silencing of EWS-FLI1 results in the exchange of EWS-FLI1 induced, activating E2F3 by constitutively expressed repressive E2F4 on the promoters of there jointly regulated target genes. Importantly, testing E2F3 promoter occupancy on wildtype and ETS motif mutated promoter constructs in ESFT cell lines revealed that binding of E2F3 to its target promoters is dependent on an intact ETS binding site. Mutation of the ETS motif resulted in reduced binding of E2F3. These data are consistent with a model in which EWS-FLI1 actively recruits an activating E2F factor thereby replacing a repressing E2F factor and in which EWS-FLI1 binding is essential for E2F binding. Strikingly, the functional E2F/ETS transcriptional module detected in ESFT for the chimeric ETS factor EWS-FLI1 was found to be also relevant to TMPRSS2-ERG expressing prostate cancer cells, but not active in HeLa cells. Supported by grants from the Austrian Science Fund (P22328-B09), and the European Comission (EU-FP7 STREP 259348). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. 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 2198. doi:1538-7445.AM2012-2198

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly binds 50% of E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves found to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1/E2F transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses and ChIP-PCR. The promoter regions of ten candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all ten genes showed reduced levels upon knockdown of EWS-FLI1. By chromatin immunoprecipitation, we confirmed that EWS-FLI1 directly binds to the promoter regions. For some of those genes, mutation of only a single ETS binding site leads to a reduction of luciferase activity comparable to knockdown of EWS-FLI1 (for example the E2F promoters). For the other selected EWS-FLI1 target candidates, mutation analysis and ChIP-PCR revealed more complex regulatory patterns. In one example, simultaneous mutation of two ETS binding sites within the region directly bound by EWS-FLI1 resulted in a significant reduction of transcriptional activity. However, mutation of the single E2F binding site rendered the promoter completely insensitive to EWS-FLI1 regulation. Furthermore, ChIP-PCR revealed that upon EWS-FLI1 repression an EWS-FLI1 induced activating E2F factor is exchanged for a repressing E2F factor on the promoter of this gene. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control shared target genes. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5341. doi:10.1158/1538-7445.AM2011-5341

The EWS-FLI1 chimeric transcription factor characteristic of Ewing's sarcoma family tumors (ESFT) constitutes the prototype of an aberrant ETS transcription factor causally involved in oncogenesis. However, the mechanisms of transcriptional regulation leading to ETS driven tumorigensis are poorly understood. Previously, we have successfully established and tested a research plan that proceeds from genomics data production, via bioinformatic generation of specific hypotheses to functional elucidation of EWS-FLI1 associated transcription network modules. In-silico analysis of time-resolved expression data revealed enrichment of ETS binding motifs in early EWS-FLI1 activated target genes, while EWS-FLI1 repressed genes showed most prominently enrichment of recognition motifs for forkhead box (FOX) proteins. Other motifs significantly co-occurring in the same promoter with FOX motifs were mainly containing homeobox domains (POU, SOX, NKX, CDP, TGIF). Concentrating on the EWS-FLI1 repressed transcriptional network most FOX proteins (FOXO1, FOXO3, FOXD1 and FOXN3) in the data set were found to be EWS-FLI1 repressed. Promoters of two of these genes, FOXO1 and FOXO3, were directly bound by EWS-FLI1 in a ChIP-seq experiment, and were also expressed at lower levels in ESFT than in mesenchymal progenitor cells which constitute a likely cell of origin for ESFT. Furthermore, bioinformatic analysis showed that FOX motifs were significantly co-enriched with ETS motifs in promoters of the same gene for ∼4% of all genes. Thus, we hypothesize that EWS-FLI1 exerts a major part of its repressive effect via inhibition of FOX proteins and may also directly (co-)repress a subset of these genes. In depth dissection of this newly identified repressive module of the EWS-FLI1 transcription network is performed by ectopic expression, RNA interference (RNAi) and reporter gene technologies combined with mutation analysis, and chromatin immunoprecipitation and protein co-immunoprecipitation. This study was funded by: “European Embryonal Tumor Pipeline” 6th framework program of the European Commission, (STREP “E.E.T. Pipeline” contract LSHC-CT-2006-037260), and by funds from the Austrian Research Fund FWF. 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 2009.

Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone and characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly regulates E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves inferred to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1 transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses. The promoter regions of nine candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all 9 genes showed reduced levels upon knockdown of EWS-FLI1. The strongest modulatory effect of EWS-FLI1 silencing was observed for the E2F3 promoter, and mutation of a single ETS binding site lead to a comparable reduction of luciferase activity as knockdown of EWS-FLI1. These results validate E2F3 as a directly EWS-FLI1 regulated gene in Ewing's sarcoma. For the other EWS-FLI1 target candidates mutation analysis lead to more complex results. In the RAD51 promoter simultaneous mutation of two ETS binding sites was necessary to obtain a significant reduction of luciferase activity. Furthermore, mutation of a single E2F binding site abolished the effect of EWS-FLI1 knockdown. In promoters of two other candidates, GEMIN4 and ATAD2, mutation of the ETS site resulted in only a moderate reduction of reporter activity, and additional destruction of an E2F binding site was required to achieve a significant decrease in promoter activity. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control common target genes. First, EWS-FLI1 directly activates E2F3 and other E2F factors which, in turn, co-operate with EWS-FLi1 on target gene promoters in a feed-forward loop. Second, results for the RAD51 promoter are compatible with a mechanism where a repressive E2F factor is exchanged for an activating E2F factor via binding of EWS-FLI1. Finally, EWS-FLI1 might have a more indirect role in the regulation of ATAD2 and GEMIN4 where most of the transcriptional effect seems to be exerted by E2F factors. Acknowledgement: This study was funded by the 6th framework program of the European Commission, (“E.E.T. Pipeline” contract LSHC-CT-2006-037260) and by the Austrian genome research program “GEN-AU Ch.I.L.D.“ (GZ 200.136/1 - VI/1/2005). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. 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 4968.

The EWS-FLI1 chimeric protein is a potent tumor specific transcriptional regulator and the molecular cause of Ewing's sarcoma family of tumors (ESFT). We had previously hypothesized that, since EWS-FLI1 is the major driver of aberrant gene expression in ESFT, there should be an inverse correlation between the changes in gene expression observed in ESFT cell lines in response to RNAi-mediated EWS-FLI1 knockdown and the corresponding gene expression in primary tumors relative to the tissue of origin. We found that mesenchymal progenitor cells (MPC) fit this assumption best and established an ESFT specific EWS-FLI1 transcriptional signature on the mRNA level. Following the same approach, we now studied the EWS-FLI1 dependent regulation of microRNAs (miRNAs) in ESFT. Genome-wide miRNA analysis was performed after shRNA-mediated stable (5 cell lines) and inducible (1 cell line) EWS-FLI1 knockdown, as well as in 6 primary ESFT and 6 MPC samples using the stem-loop reverse transcription quantitative PCR platform. Among miRNAs consistently affected by EWS-FLI1 silencing and inversely expressed in primary tumors compared to MPCs was hsa-miR-145 which we found upregulated upon EWS-FLI1 silencing. Since several target sites exist for this miRNA within the FLI1 3′UTR, we asked if hsa-miR-145 is capable of modulating EWS-FLI1 expression in ESFT cells. Upon ectopic expression of pri-miR-145 in wildtype p53 ESFT cells, strong modulation of EWS-FLI1 protein levels, but not RNA levels, was observed. In p53 mutant cell lines this effect was less pronounced consistent with a recently reported role of p53 in miR-145 maturation. Preliminary studies suggest that hsa-miR-145 is indirectly regulated by EWS-FLI1 by a mechanism that may involve the NOTCH signaling pathway. Our results identify a positive feed-back loop between EWS-FLI1 and hsa-miR-145 expression in ESFT. Supported by the 6th framework program of the European Commission, (STREP “E.E.T. Pipeline“ contract LSHC-CT-2006-037260) and grant P20665-B12 of the Austrian Science Fund FWF. 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 1949.

The polymerase chain reaction (PCR) was applied to the problem of rhinoviral serotyping and the detection of mutations in large DNA fragments. For serotyping, a region of about 380 nucleotides from the 5’-untranslated region of the genomes of six human rhinovirus serotypes was amplified by the PCR using one pair of primers. The DNAs were cleaved with selected restriction enzymes, and the fragments were analyzed on polyacrylamide gels. The cleavage patterns enabled the characterization of the different serotypes. For the detection of mutations, cDNAs corresponding to the whole capsid region of rhinovirus variants stable at low pH were amplified. Restriction fragments of the amplified DNAs were analyzed on two-dimensional gels, separating in the first dimension according to size and in the second dimension according to the secondary structure of the single strands. When wild type and mutants were compared, a different migration of the single strands from two fragments was observed. The fragments of interest were eluted from the gel and directly sequenced. The method presented thus allows rapid identification of the location and of the exact nature of point mutations.