Ewing sarcoma is an aggressive bone cancer of children and young adults defined by the presence of a chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the amino-terminal domain of EWS to the carboxyl-terminus of FLI. The EWS portion is an intrinsically disordered transcriptional regulatory domain, while the FLI portion contains an ETS DNA-binding domain and two flanking regions of unknown function. Early studies using non-Ewing sarcoma models provided conflicting information on the roles of each domain of FLI in EWS/FLI oncogenic function. We therefore sought to define the specific contributions of each FLI domain to EWS/FLI activity in a well-validated Ewing sarcoma model and, in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. We analyzed a series of engineered EWS/FLI mutants with alterations in the FLI portion using a variety of assays. Fluorescence anisotropy, CUT&RUN, and ATAC-sequencing experiments revealed that the isolated ETS domain is sufficient to maintain the normal DNA-binding and chromatin accessibility function of EWS/FLI. In contrast, RNA-sequencing and soft agar colony formation assays revealed that the ETS domain alone was insufficient for transcriptional regulatory and oncogenic transformation functions of the fusion protein. We found that an additional alpha-helix immediately downstream of the ETS domain is required for full transcriptional regulation and EWS/FLI-mediated oncogenesis. These data demonstrate a previously unknown role for FLI in transcriptional regulation that is distinct from its DNA-binding activity. This activity is critical for the cancer-causing function of EWS/FLI and may lead to novel therapeutic approaches.

Purpose Neuroblastoma is an embryonic childhood cancer with high mortality. 13-cis retinoic acid (13-cisRA) improves survival for some patients, but many recur, suggesting clinical resistance. The mechanism of resistance, and the normal differentiation pathway, are poorly understood. Three-Amino-acid Loop Extension (TALE) family genes are master regulators of differentiation. Since retinoids promote differentiation in neuroblastoma, we evaluated TALE family gene expression in neuroblastoma. Experimental Design We evaluated expression of TALE family genes in RA-sensitive and -resistant neuroblastoma cell lines, with and without 13cis-RA treatment, identifying genes whose expression correlate with retinoid sensitivity. We evaluated the roles of one gene, PBX1, in neuroblastoma cell lines, including proliferation and differentiation. We evaluated PBX1 expression in primary human neuroblastoma samples by RT-qPCR, and three independent clinical cohort microarray datasets. Results We confirmed induction of PBX1 expression, and no other TALE family genes, was associated with 13-cisRA responsiveness in NB cell lines. Exogenous PBX1 expression in neuroblastoma cell lines, mimicking induced PBX1 expression, significantly impaired proliferation and anchorage-independent growth, and promoted RA-dependent and -independent differentiation. Reduced PBX1 protein levels produced an aggressive growth phenotype and RA resistance. PBX1 expression correlated with histological neuroblastoma subtypes, with highest expression in benign ganglioneuromas and lowest in high-risk neuroblastomas. High PBX1 expression is prognostic of survival, including in multivariate analysis, in the three clinical cohorts. Conclusions PBX1 is an essential regulator of differentiation in neuroblastoma and potentiates retinoid-induced differentiation. Neuroblastoma cells and tumors with low PBX1 expression have an immature phenotype with poorer prognosis, independent of other risk factors.

Paediatric cancers commonly harbour quiet mutational landscapes and are instead characterized by single driver events such as the mutation of critical chromatin regulators, expression of oncohistones, or expression of oncogenic fusion proteins. These events ultimately promote malignancy through disruption of normal gene regulation and development. The driver protein in Ewing sarcoma, EWS/FLI, is an oncogenic fusion and transcription factor that reshapes the enhancer landscape, resulting in widespread transcriptional dysregulation. Lysine-specific demethylase 1 (LSD1) is a critical functional partner for EWS/FLI as inhibition of LSD1 reverses the transcriptional activity of EWS/FLI. However, how LSD1 participates in fusion-directed epigenomic regulation and aberrant gene activation is unknown. We now show EWS/FLI causes dynamic rearrangement of LSD1 and we uncover a role for LSD1 in gene activation through colocalization at EWS/FLI binding sites throughout the genome. LSD1 is integral to the establishment of Ewing sarcoma super-enhancers at GGAA-microsatellites, which ubiquitously overlap non-microsatellite loci bound by EWS/FLI. Together, we show that EWS/FLI induces widespread changes to LSD1 distribution in a process that impacts the enhancer landscape throughout the genome.

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or a highly related FET/ETS chimera. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma cells using a well-validated ‘knock-down/rescue’ model of EWS/FLI function in combination with next generation sequencing assays to evaluate how the chromatin landscape changes with loss, and recovery, of EWS/FLI expression. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. This EWS/FLI binding signature is associated with establishment of topologically-associated domain (TAD) boundaries, compartment activation, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. In addition, EWS/FLI co-localizes with the loop-extrusion factor cohesin to promote chromatin loops and TAD boundaries. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators of three-dimensional reprogramming of chromatin.

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly “undruggable” therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor I-BET151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in four models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of I-BET151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that I-BET151 and alisertib are effective in prolonging survival in four xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.

Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS–FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS–FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.

Ewing sarcoma is a pediatric bone cancer defined by a chromosomal translocation fusing one of the FET family members to an ETS transcription factor. There have been seven reported chromosomal translocations, with the most recent reported over a decade ago. We now report a novel FET/ETS translocation involving FUS and ETV4 detected in a Ewing sarcoma patient.Here, we characterized FUS/ETV4 by performing genomic localization and transcriptional regulatory studies on numerous FET/ETS fusions in a Ewing sarcoma cellular model. Through this comparative analysis, we demonstrate significant similarities across these fusions, and in doing so, validate FUS/ETV4 as a bona fide Ewing sarcoma translocation. This study presents the first genomic comparison of Ewing sarcoma-associated translocations and reveals that the FET/ETS fusions share highly similar, but not identical, genomic localization and transcriptional regulation patterns. These data strengthen the notion that FET/ETS fusions are key drivers of, and thus pathognomonic for, Ewing sarcoma. ImplicationsIdentification and initial characterization of the novel Ewing sarcoma fusion, FUS/ETV4, expands the family of Ewing-fusions and extends the diagnostic possibilities for this aggressive tumor of adolescents and young adults.

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly "undruggable" therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor IBET-151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in three models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of IBET-151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that IBET-151 and alisertib are effective in prolonging survival in three xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.