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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. Independent but complementary studies from Vakoc and Stegmaier identify and characterize a role for ETV6 in counteracting the transcriptional activity of EWS–FLI during Ewing sarcoma development, which may be targeted for therapeutic benefits.

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.

OBJECTIVES/SPECIFIC AIMS: (1) Correlate PBX1 mRNA expression as measured by RNAScope in situ hybridization, at an RNA number/cell measurement, Versus by RT-qPCR by the ddCt method. (2) Validate PBX1 mRNA expression in a second independent cohort of neuroblastoma tumor samples, and correlate with patient outcomes. We expect that PBX1 expression will correlate whether detected by RNAScope or by RT-qPCR. This work has the promise of validating a novel biomarker of disease severity, and for clinical translation as the RNAscope technology has been CLIA-certified for clinical use for other genes. METHODS/STUDY POPULATION: Primary neuroblastoma tumor samples were acquired through the Children’s Oncology Group Tumor Bank, The Cooperative Human Tissue Network Tumor bank, and the Westmeade Tumor Bank (Westmeade, Australia), with patient outcomes annotated but sequestered until experiments are completed. RT-qPCR is performed using 1 μg total RNA isolated from each sample by Nucleospin RNA kit (Clontech), reverse transcribed by SuperScript VILO (ThermoFisher Scientific) and amplified using KiCqSTART SYBR Green qPCR mix (Sigma Aldrich). RNAScope was performed on sections of fresh frozen tumor, in triplicate, per manufacturer protocol (ACDBio) using company-designed probes. Statistical analyses performed using GraphPad Prism5. RESULTS/ANTICIPATED RESULTS: PBX1 mRNA expression as measured RNAScope correlated well with matched RT-qPCR values, with most PBX1 transcripts identified within the malignant cells and not in tumor stroma. Correlation with patient outcomes is ongoing (expected to be available at the time of presentation), but as the RNAScope values correlate with R>0.9 with RT-qPCR values, we expect good correlation with outcomes in our primary data set and matching validation set. DISCUSSION/SIGNIFICANCE OF IMPACT: PBX1 mRNA expression is an accurate prognostic biomarker of outcome in low and intermediate-risk neuroblastoma, and testing on an additional validation set is planned based on thresholds established by RNAScope. RNAScope is a method readily translatable to clinical use and its inclusion in future clinical trials will be further studied. It provides an additional benefit that concomitant immunohistochemistry can also be performed. Analysis of high-risk neuroblastomas for responsiveness to retinoic acid based on PBX1 expression is planned.

OBJECTIVES/SPECIFIC AIMS: (1) Correlate PBX1 mRNA expression as measured by RNAScope in situ hybridization, at an RNA number/cell measurement, Versus by RT-qPCR by the ddCt method. (2) Validate PBX1 mRNA expression in a second independent cohort of neuroblastoma tumor samples, and correlate with patient outcomes. We expect that PBX1 expression will correlate whether detected by RNAScope or by RT-qPCR. This work has the promise of validating a novel biomarker of disease severity, and for clinical translation as the RNAscope technology has been CLIA-certified for clinical use for other genes. METHODS/STUDY POPULATION: Primary neuroblastoma tumor samples were acquired through the Children’s Oncology Group Tumor Bank, The Cooperative Human Tissue Network Tumor bank, and the Westmeade Tumor Bank (Westmeade, Australia), with patient outcomes annotated but sequestered until experiments are completed. RT-qPCR is performed using 1 μg total RNA isolated from each sample by Nucleospin RNA kit (Clontech), reverse transcribed by SuperScript VILO (ThermoFisher Scientific) and amplified using KiCqSTART SYBR Green qPCR mix (Sigma Aldrich). RNAScope was performed on sections of fresh frozen tumor, in triplicate, per manufacturer protocol (ACDBio) using company-designed probes. Statistical analyses performed using GraphPad Prism5. RESULTS/ANTICIPATED RESULTS: PBX1 mRNA expression as measured RNAScope correlated well with matched RT-qPCR values, with most PBX1 transcripts identified within the malignant cells and not in tumor stroma. Correlation with patient outcomes is ongoing (expected to be available at the time of presentation), but as the RNAScope values correlate with R >0.9 with RT-qPCR values, we expect good correlation with outcomes in our primary data set and matching validation set. DISCUSSION/SIGNIFICANCE OF IMPACT: PBX1 mRNA expression is an accurate prognostic biomarker of outcome in low and intermediate-risk neuroblastoma, and testing on an additional validation set is planned based on thresholds established by RNAScope. RNAScope is a method readily translatable to clinical use and its inclusion in future clinical trials will be further studied. It provides an additional benefit that concomitant immunohistochemistry can also be performed. Analysis of high-risk neuroblastomas for responsiveness to retinoic acid based on PBX1 expression is planned.

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI1, DBD-𝛼4 helix, is critical for the transcription function of EWSR1::FLI1. In this study, we aimed to understand the mechanism by which the DBD-𝛼4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatinmarks, genome binding, and gene expression in cells expressing EWSR1::FLI1 constructs with and without the DBD-𝛼4 helix. Our studies revealed DBD-𝛼4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

Genes encoding the RNA-binding proteins FUS, EWSR1, and TAF15 (FET proteins) are involved in chromosomal translocations in rare sarcomas. FET-rearranged sarcomas are often aggressive malignancies affecting patients of all ages. New therapies are needed. These translocations fuse the 5' portion of the FET gene with a 3' partner gene encoding a transcription factor (TF). The resulting fusion proteins are oncogenic TFs with a FET protein low complexity domain (LCD) and a DNA binding domain. FET fusion proteins have proven stubbornly difficult to target directly and promising strategies target critical co-regulators. One candidate is lysine specific demethylase 1 (LSD1). LSD1 is recruited by multiple FET fusions, including EWSR1::FLI1. LSD1 promotes EWSR1::FLI1 activity and treatment with the noncompetitive inhibitor SP-2509 blocks EWSR1::FLI1 transcriptional function. A similar molecule, seclidemstat (SP-2577), is currently in clinical trials for FET-rearranged sarcomas (NCT03600649). However, whether seclidemstat has pharmacological activity against FET fusions has not been demonstrated. Here, we evaluate the potency of seclidemstat against multiple FET-rearranged sarcoma cell lines, including Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, and myxoid liposarcoma. We also define the transcriptomic effects of seclidemstat treatment and evaluated the activity of seclidemstat against FET fusion transcriptional regulation. Seclidemstat showed potent activity in cell viability assays across FET-rearranged sarcomas and disrupted the transcriptional function of all tested fusions. Though epigenetic and targeted inhibitors are unlikely to be effective as a single agents in the clinic, these data suggest seclidemstat remains a promising new treatment strategy for patients with FET-rearranged sarcomas.

Ewing sarcoma is the second most common bone cancer in children and young adults. In ~85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWS::FLI. EWS::FLI is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWS portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWS::FLI. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI, DBD-a4 helix, is critical for the transcription function of EWS::FLI. In this study, we aimed to understand the mechanism by which the DBD-a4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWS::FLI constructs with and without DBD-a4 helix. Our studies revealed DBD-a4 helix is crucial for cooperative binding of EWS::FLI at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWS::FLI such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.Competing Interest StatementThe authors have declared no competing interest.

Ewing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or highly related fusions. 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. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. EWS/FLI binding is primarily associated with compartment activation, establishment of topologically-associated domain (TAD) boundaries, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. 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 through 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 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.

Pediatric cancers commonly harbor 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. Competing Interest Statement There are three conflicts-of-interest to disclose: Stephen Lessnick is on the scientific advisory board for, and Sunil Sharma is a founder and equity holder of, Salarius Pharmaceuticals; currently undertaking clinical development of LSD1 inhibitors for Ewing sarcoma. Stephen Lessnick is also listed as an inventor on United States Patent No. US 7,939,253 B2, \"Methods and compositions for the diagnosis and treatment of Ewing's Sarcoma\" and United States Patent No. US 8,557,532, \"Diagnosis and treatment of drug-resistant Ewing's sarcoma.\" These are not affected by the data reported in this report and do not impact our adherence to bioRxiv policies on sharing data and materials.