EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma

Abstract

Purpose: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. Experimental Design: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. Results: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. Conclusions: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13.

EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma

“…With the inclusion of EWS/FLI mediated chromatin loops, we identified 193 genes that are dysregulated by a conserved EWS/FLI peak associated with a gained loop in EF-Endo versus EF-KD. These include genes that were previously shown to be EWS/FLI responsive and play important roles in tumor growth and/or metastasis in Ewing sarcoma, such as NKX2-2 ( 8 , 15 ), PPP1R1A ( 70 ), HOXD13 ( 71 ), FEZF1 ( 11 , 72 ), CCND1 ( 15 , 73 ) and CAV1 ( 74 ). We also identified target genes that code for surface proteins, such as SLC24A3 , NTNG1 , LINGO1 and PAPPA , reported as potential candidates for antibody-mediated therapies in Ewing sarcoma ( 75 , 76 ).…”

The importance of fusion protein activity in Ewing sarcoma and the cell intrinsic and extrinsic factors that regulate it: A review

“…These studies have shown that expression and transcriptional activity of the fusion varies among tumor cells and that cells exist along a transcriptional continuum from less to more mesenchymal states. Significantly, this heterogeneity is evident in tumor-derived cell lines and patient-derived xenografts (PDX) in vitro and in vivo and in patient tumor biopsies ( 17 , 22 , 33 , 34 ). Consistent with genetic knockdown studies, cells with higher EWS::FLI1 activity express proliferative signatures, while cells with lower transcriptional activity upregulate mesenchymal gene signatures and display enhanced metastatic potential ( 17 , 22 , 33 ).…”

“…Significantly, this heterogeneity is evident in tumor-derived cell lines and patient-derived xenografts (PDX) in vitro and in vivo and in patient tumor biopsies ( 17 , 22 , 33 , 34 ). Consistent with genetic knockdown studies, cells with higher EWS::FLI1 activity express proliferative signatures, while cells with lower transcriptional activity upregulate mesenchymal gene signatures and display enhanced metastatic potential ( 17 , 22 , 33 ). Elucidating the contribution of these distinct cell states to local and metastatic progression, treatment response, and relapse is an area of intense investigation in the field.…”

“…Reduction of the fusion leads to decreased expression of EWS::FLI1-activated targets and increased expression of repressed targets. However, the mechanisms by which the fusion activates and represses target genes are distinct and, although significant gaps in knowledge still exist, it is evident that the activation and repressive signatures can be dissociated depending on the presence of additional regulators of these separate processes ( 20 – 22 ). Below we summarize the current understanding of how EWS::FLI1 alters gene transcription.…”

“…Other transcription factors can instead counter-balance EWS::FLI1 activity. The developmental limb patterning TF HOXD13, for example, is upregulated by EWS::FLI1 but induces mesenchymal gene programs that are repressed by EWS::FLI1 ( 22 ). HOXD13 therefore partly antagonizes EWS::FLI1 function, and the competing activities of these TFs determine transcriptional cell states along a mesenchymal axis ( 22 ).…”