A Druggable Rheostat for Ewing Sarcoma?

In this issue of Clinical Cancer Research, Apfelbaum and colleagues define a spectrum of mesenchymal states in Ewing tumor cells and demonstrate the novel regulation of Ewing cell mesenchymal plasticity by both the fusion oncoprotein EWS::FLI1 and the developmental transcription factor HOXD13 (1). First, the regulation of HOXD13 expression by EWS::FLI1 is methodically dissected. The authors pinpoint an EWS::FLI1-dependent enhancer (which they term the “posterior HOXD enhancer,”) that controls HOXD13 expression. Subsequently, the ramifications of HOXD13 expression on Ewing tumor cells is examined, and they observe that HOXD13 represses neuronal differentiation signatures while promoting cell adhesion programs. A modest reduction in cell motility upon HOXD13 expression loss is also noted. Quite interestingly, through the use of CUT&RUN sequencing and single-cell analyses, the authors find that although EWS::FL1 participates in the regulation of HOXD13 expression, once expressed, HOXD13 actually promotes an antagonistic transcriptional signature to that of EWS::FLI1. Specifically, HOXD13 enhances the transcription of genes normally repressed by EWS::FLI1. The authors thus dub HOXD13 a “rheostat” for EWS::FLI1 transcriptional activity.

The importance of EWS::FLI1 level on Ewing cell behavior is evident in the field. Ewing sarcoma cells with “high” EWS::FLI1 expression tend to possess a proliferative phenotype while cells with “low” EWS::FLI1 expression are highly migratory and/or invasive yet proliferate more slowly (2, 3). EWS::FLI1 high versus low cell states result from either changes in the total expression of the fusion oncoprotein and/or modifiers disrupting EWS::FLI1 transcriptional programs. In the current work, Apfelbaum and colleagues clearly demonstrate that HOXD13 modulation of Ewing cell behavior occurs through transcriptional upregulation of EWS::FLI1 repressed genes and not through modulation of fusion oncoprotein expression. Pedersen and colleagues previously demonstrated the ability of Wnt pathway activation to shift Ewing cells to a more metastatic EWS::FLI1 “low” state (4). Very recently, Seong and colleagues described the ability of TRIM8 to degrade the EWS::FLI1 fusion oncoprotein and serve as a key regulator of EWS::FLI1 expression (5). This work and others note that too much or too little EWS::FLI1 is toxic to Ewing sarcoma cells, thus providing potential therapeutic opportunities through EWS::FLI1 targeting (6).

When considering HOXD13 expression in Ewing sarcoma through a therapeutic lens, three key questions arise: (i) Is HOXD13 expression (and thus the Ewing mesenchymal cell state) impacted by chemotherapy?, (ii) As Apfelbaum and colleagues note in their discussion of the current work, can Ewing cells shift between HOXD13 high and low activity states?, and (iii) Can simultaneously targeting multiple cell states result in more effective treatments for patients with Ewing sarcoma?

HOXD13 activation was noted by Apfelbaum and colleagues not only in vitro but also in patient-derived xenografts and primary Ewing tumors from patients. As a precursor to thinking about an approach to targeting HOXD13 high Ewing cell populations in the future, understanding whether tumor status impacts the prevalence of HOXD13 “high” tumor cells is key. Utilizing paired patient samples to examine HOXD13 expression and/or HOXD13 transcriptional signatures at the time of diagnosis (pretherapy) and during/posttherapy (local control, disease progression, or disease relapse) would help guide future preclinical studies aimed at targeting this tumor cell population. In addition, understanding whether specific treatments, especially agents utilized in the setting of disease relapse (radiation, irinotecan, temozolamide, topotecan, etc.), impact HOXD13 expression and the mesenchymal state of Ewing tumor cells is an important preclinical question to address.

A related question, and one posed by the authors in the discussion, is whether Ewing cells are held “static” in HOXD13 activity states or whether cells can rapidly shift between high and low HOXD13 activity? Should Ewing cells harbor the ability to transition between the HOXD13 activity states outlined in the current article, determining the mechanism regulating this plasticity may provide clues to targeting cells during this transition. Hypothetically, if a drug was known to specifically eliminate a population of HOXD13 high activity cells (as compared with a low HOXD13 state), inhibiting cells from transitioning or forcing them into a HOXD13 high state could be a novel approach to improve overall treatment efficacy.

Additional translational potential of this work includes determining whether simultaneous targeting of multiple Ewing cell states (e.g., HOXD13 “high” cells and EWS::FLI1 “high” cells) could more effectively treat Ewing tumors (Fig. 1). The authors demonstrate in patient primary tumors and Ewing PDX samples that HOXD13 expression and activity demonstrate intercellular variability. In addition, the data presented by Apfelbaum and colleagues provide clues to potential approaches for targeting distinct HOXD13 cell populations in the future. Protein expression of CD73 is used in this work as a marker of HOXD13 high cells (a mesenchymal cell state). CD73 is encoded by the gene NT5E (also demonstrated to be impacted by HOXD13 in this work) and functions to convert AMP to adenosine. As adenosine promotes immunosuppression in the tumor microenvironment, CD73 expression is a potential immunotherapeutic target (7, 8). While CD73 was used in this work to identify mesenchymal cell populations, given its role in the immune microenvironment, CD73 could be investigated as a potential immunotherapeutic target to negative the immunosuppressive role of HOXD13 high cells. Alternatively, given the rise in the generation and use of antibody drug conjugates for the treatment of cancer, CD73 could be investigated as a target to deliver directed therapy against this CD73 high Ewing cell population.

In addition to CD73, another potential avenue for targeting HOXD13 high cells is revealed in the author's RNA sequencing (RNA-seq) data examining shNS versus shHOXD13. It is known that EWS::FLI1 downregulates the TGFβ receptor in Ewing sarcoma (9). The current work demonstrates that HOXD13 antagonizes this transcriptional downregulation, thus HOXD13 high cells may be a subpopulation of Ewing sarcoma cells that express the TGFβ receptor (TGFBR2). Indeed, the RNA-seq findings suggest that HOXD13 high Ewing sarcoma cells express more TGFBR2 than cells with low HOXD13 activity. Protein expression of TGFBR2 would need to be determined to validate this as a potential drug target. Like CD73, TGFβ signaling can also promote immunosuppression in the tumor microenvironment, thus making chemotherapy and/or immunotherapy combinations of potential interest when considering concomitant targeting of rapidly proliferating EWS::FLI1 high/HOXD13 low cells and mesenchymal, immunosuppressive EWS::FLI1 low/HOXD13 high cell populations.

Patients with aggressive Ewing sarcoma need fresh, biologically guided treatment approaches aimed at improving outcomes. Apfelbaum and colleagues provide an exciting advancement of our understanding of the heterogeneity and plasticity of Ewing sarcoma cells. Preclinically testing therapies aimed at targeting >1 cell state and/or targeting cell state transitions is a needed next step in the field.

No disclosures were reported.

K.M. Bailey is supported by the NCI (1K08CA252178) and by a Hyundai Hope on Wheels Young Investigator award. K.M. Bailey would also like to thank the UPMC Children's Hospital Foundation. K.R. Weiss is supported by the Make It Better Foundation, the Shadyside Hospital Foundation, the Connective Tissue Oncology Society, and Pittsburgh Cure Sarcoma.