EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors

IGF1 Is a Common Target Gene of Ewing's Sarcoma Fusion Proteins in Mesenchymal Progenitor Cells

The EWS-FLI-1 fusion protein is associated with 85-90% of Ewing's sarcoma family tumors (ESFT), the remaining 10-15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3-5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis

Epigenetic Features of Human Mesenchymal Stem Cells Determine Their Permissiveness for Induction of Relevant Transcriptional Changes by SYT-SSX1

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects

R1507, an Anti-Insulin-Like Growth Factor-1 Receptor (IGF-1R) Antibody, and EWS/FLI-1 siRNA in Ewing's Sarcoma: Convergence at the IGF/IGFR/Akt Axis

A subset of patients with Ewing's sarcoma responds to anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies. Mechanisms of sensitivity and resistance are unknown. We investigated whether an anti-IGF-1R antibody acts via a pathway that could also be suppressed by small interfering (si) RNA against the EWS/FLI-1 fusion protein, the hallmark of Ewing's sarcoma. The growth of two Ewing's sarcoma cell lines (TC-32 and TC-71) was inhibited by the fully human anti-IGF-1R antibody, R1507 (clonogenic and MTT assays). TC-32 and TC-71 cells express high levels of IGF-2, while RD-ES and A4573 Ewing's cell lines, which were less responsive to R1507 in our assays, express low or undetectable IGF-2, respectively. TC-71 cells also expressed high levels of IGF-1R, and R1507 decreased steady-state levels of this receptor by internalization/degradation, an effect which was associated with a decrease in p-IGF-1R, p-IRS-1, and p-Akt. EWS/FLI-1 siRNA also decreased p-Akt, due to its ability to increase IGF-BP3 levels and subsequently decrease IGF-1 and IGF-2 levels, thus inhibiting signaling through p-IGF-1R. This inhibition correlated with growth suppression and apoptosis. The attenuation of Akt activation was confirmed in TC-71 and HEK-293 (human embryonic kidney) cells by transfecting them with IGF-1R siRNA. We conclude that antibodies and siRNA to IGF-1R, as well as siRNA to EWS/FLI-1, act via intersecting IGF/IGF-1R signals that suppress a common point in this pathway, namely the phosphorylation of Akt

High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy

Diacylglycerol lipase activation and 5-lipoxygenase activation and translocation following TCR/CD3 triggering in T cells.

Arachidonic acid (AA) release was observed following T cell receptor (TCR)/CD3 complex cross-linking in different tumor T cell lines as well as on purified peripheral T cells in vivo. Direct measurement of enzymatic activity in vitro of TCR/CD3-stimulated Jurkat cell extracts on labeled vesicle substrates showed that TCR/CD3 cross-linking resulted in AA release from sn-1,2-diacylglycerol (DAG) vesicles, as detected by TLC analysis, suggesting that DAG lipase was activated following TCR/CD3 stimulation and DAG generation. On the contrary, no phospholipase A2 activation was observed in response to TCR/CD3 stimulation, since no lyso-phospholipids were generated in vitro from either phosphatidylcholine or phosphatidylinositol-3,4-bisphosphate, or from phosphatidic acid vesicles. Moreover, the 1-DAG lipase inhibitor RHC80267 completely blocked TCR/CD3-dependent AA release in vitro and in vivo, without effect upon TCR/CD3-dependent inositol-1,4,5-trisphosphate (IP3) generation. Importantly, evidence for further metabolism of released AA was obtained, since synthesis and release of cysteinyl leukotrienes (CLT), but not of leukotriene B4 or cyclooxygenase products, could be detected by radioimmunoassay in different T cell lines and peripheral blood T cells following TCR/CD3 cross-linking. Moreover, HPLC analysis revealed an accumulation of leukotriene E4 in TCR/CD3 stimulated Jurkat cells. This was associated with translocation of 5-lipoxygenase from the cytosol to the cell membranes. Finally, TCR/CD3-mediated CLT production was blocked by MK886, a specific inhibitor of 5-LO translocation and activation. Our data help define a further level in the fate of second messengers generated after TCR/CD3 triggering and suggest that additional mediators can play a role in the context of T cell activation

Follicle-stimulating hormone induced-phospholipase A2 activity and -eicosanoid generation in rat Sertoli cells.

The possibility that FSH stimulates the phospholipase A(2) (PLA(2)) pathway was studied in cultured immature Sertoli cells. FSH induced [H-3]-arachidonic acid (AA) release from prelabeled cells in a time- and concentration-dependent fashion (ED(50) = 21.8 +/- 1.9 ng/ml). This response could be fully prevented by pretreatment of cells with the PLA(2) inhibitor, mepacrine. That PLA(2) was the main enzyme responsible for cleavage of AA from membrane phospholipids was directly shown by PLA(2) activity assay using vesicles of radiolabeled phosphatidylcholine (PC) as substrate. Furthermore, FSH stimulated eicosanoid generation in a time-dependent manner through the cyclooxygenase but not the lipoxygenase pathway. In fact, higher levels of prostaglandin (PG) E(2), F-2 alpha,F- and the stable products of PGI(2) and thromboxane A(2) (6-keto PGF(1 alpha) and thromboxrane B-2, respectively) were generated by the gonadotropin-treated cells as compared to control cells. The effect was inhibited by mepacrine, further supporting the pivotal role of PLA(2) in the release of the eicosanoid precursor, AA. Finally, the effect of the main product of FSH-induced AA metabolism, i.e., PGE(2), was studied. Intracellular cAMP accumulation in Sertoli cells was stimulated by the prostanoid in a dose-dependent manner (ED(50) = 2.3 +/- 0.37 nM). PGE(2) also significantly stimulated aromatase activity, a specific marker of Sertoli cell functions, measured as 17 beta-estradiol production (ED(50) = 4.7 +/- 0.8 nM). Similar results were obtained with PGF(2 alpha). Our findings show that FSH, through the activation of PLA(2), leads to AA release with consequent metabolism by the cyclooxygenase pathway. Prostanoids produced by Sertoli cells upon FSH stimulation can control, in an autocrine or paracrine manner, the functions of the somatic cells in the seminiferous epithelium