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 chromatin marks, 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 topologically associated domains (TADs), chromatin loops, enhancers, and productive transcription hubs.

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- α 4 helix, is critical for the transcription function of EWS::FLI. In this study, we aimed to understand the mechanism by which the DBD- α 4 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- α 4 helix. Our studies revealed DBD- α 4 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.

Supplemental Table 1. Association of clinical features with BM micrometastatic cell burden (CD99+CD45- percent) in flow cytometry cohort. Supplemental Table 2. Total CD99+CD45- percent burden in Ewing sarcoma patients with clinically evident BM metastasis (N=6).

Supplementary materials, including supplementary Methods, Table S3. Primer Sequences. Figure S1. Transcriptional Profiling of HCI2509 in A673 and TTC-466. Figure S2. Morphological Changes in TTC-466 with HCI2509 Treatment. Figure S3. Effects of HCI2509 on Transformation, Methylation, and Apoptosis. Figure S4. Regulation of HMOX1. Figure S5. Tumor Volume, Body Weight, and Blood Counts.

Cell line source/culture conditions, primer/shRNA sequences, antibodies utilized in this study and additional methods

Ewing sarcoma is an aggressive bone-associated tumor currently treated with dose-intense chemotherapy, radiation, and surgery and it affects adolescents and young adults. The hallmark of Ewing sarcoma is a translocated fusion transcription factor named EWS::FLI that drives the oncogenic process. We hypothesize that FLI portion of EWS::FLI containing a crucial alpha-helix plays a novel role in transcription regulation of thousands of genes by modulating chromatin looping. The hypothesis is based on recent evidences from our lab: an alpha-helix immediately downstream of DNA binding domain as an important player in regulating transcriptional activity and that EWS::FLI has a substantial role in shaping the chromatin landscape of Ewing sarcoma cells. The objective of this study is to elucidate the mechanism underlying transcriptional regulation by FLI. We utilized knockdown/rescue experiments in which EWS::FLI was depleted with shRNA and replaced with constructs containing the ETS DNA Binding Domain (DBD) alone or DBD+ (DBD and 4th alpha-helix). The binding pattern and transcriptional regulation were assessed with CUT&Tag and RNA-Seq respectively. The extent of roles each of these construct play in organization, structure, and function of chromatin are being assessed using Micro-C technique, a variation of Hi-C with improved resolution, higher signal-to-noise ratio and more information on chromatin domain boundaries and chromatin looping. The DNA binding and genomic localization of EWS::FLI was unaltered by the deletion surrounding the DNA binding domain (which contains a 4th alpha-helix) in A673 and TTC466 cells. Despite this similarity in genomic localization and binding, the transcriptional output driven by EWS::FLI was significantly diminished by the deletion. With this current study, we hope to understand if the flanking region neighboring the DNA binding domain contributes to the chromatin architecture remodeling function of EWS::FLI and whether this function could be attributed to the transcriptional output differences in the DBD and DBD+ conditions. Citation Format: Ariunaa Bayanjargal, Cenny Taslim, Jesse Crow, Julia Selich-Anderson, Stephen L. Lessnick. Role of FLI portion of EWS::FLI in transcription regulation via modulation of chromatin 3D landscape in Ewing sarcoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3558.

Supplementary Figure from Mitochondrial Dysfunction Is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma

Supplementary Cancer Cell article reference

Supplementary Figure 2 from EWS/FLI1 Regulates Tumor Angiogenesis in Ewing's Sarcoma via Suppression of Thrombospondins

Supplementary Figure 1 from EWS/FLI1 Regulates Tumor Angiogenesis in Ewing's Sarcoma via Suppression of Thrombospondins

PDF file, 59K, Supplementary Figure S1. A typical example of Bilateral Inguinal Lymph node size 8 days after vaccination with the native peptide QQNPSYDSV (#14) or its modified derivative YLNPSVDSV (#24).

Supp Figure 8 depicts regulated pathways of the FET/ETS fusions in Ewing sarcoma cells.

Supplementary Table S2. Nine-Gene Panel qRT-PCR Validation p-values.

Protein expression of ER-stress response genes following treatment with SP-2509

Validation of KDM1A knockdown in Ewing sarcoma cells

Supplementary Figure 3 from EWS/FLI1 Regulates Tumor Angiogenesis in Ewing's Sarcoma via Suppression of Thrombospondins

Supp Figure 7 contains results of a pathway analysis performed on FET/ETS-regulated genes in Ewing sarcoma cells.

Supp Figure 4 depicts the overlap analysis of EWS/ETS and FUS/ETS proteins at both the genomic localization and transcriptional regulatory levels.

Supplementary Methods from NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma

Summary of Ewing sarcoma KDM1A expression studies and genes consistently induced/repressed following SP-2509 treatment