Abstract 5695: CyTOF analysis of targeted therapy in addition to standard of care against Ewing sarcoma tumor subpopulations

Abstract Introduction: The overall survival for Ewing sarcoma (ES) is ∼70% after current standard of care (SOC) chemotherapy but outcomes remain dismal for relapsed or metastatic disease. SOC involves chemotherapy using alternating cycles of Vincristine, Doxorubicin, Cyclophosphamide (VDC) with Ifosfamide and Etoposide (IE). ES tumors are increasingly being studied in the context of the EWS:FLI1 fusion. Recent work has shown that the EWS:FLI1high cells are more proliferative and sensitive to chemotherapy and the EWS:FLI1low cells are more invasive and less sensitive to chemotherapy. Cancer stem cells are another population known to be chemoresistant. The invasiveness and decreased sensitivity of these various populations suggest they may contribute to chemoresistance, relapse, and metastasis, representing a major clinical problem. Previous work has shown that HDAC inhibition synergizes with SOC agents due to an accumulation of DNA damage, though the effect on specific subpopulations within tumors remains unknown. The purpose of this study was to identify effects of romidepsin combined with SOC treatment on specific subpopulations of ES cells. Methods: For in vitro experiments, A673 ES cells were treated with vehicle, romidepsin, IE, or romidepsin+IE for 24 hours and then stained for CyTOF analysis. For in vivo experiments, A673 cells were injected subcutaneously into Nude mice. Once tumors were 150-200mm3, mice were grouped and treatment with vehicle, romidepsin, ifosfamide/etoposide (IE), or romidepsin+IE began. Romidepsin was given on days 1 and 4 and IE was given days 2-4. Tumors were harvested and dissociated on day 5 for CyTOF analysis. The CyTOF panel used was designed to assess tumor subpopulations as well as mechanistic changes. Results: In vivo and in vitro, the combination treatment led to a significant increase in pH2AX in the bulk tumor cells, indicative of DNA damage. In vivo, UMAP display of phenograph clustering, and associated heatmap of expression, revealed distinct changes in expression patterns among treatment groups. In the EWS:FLI1high population both IE alone and IE in combination with romidepsin led to a significant increase in pH2AX. In the EWS:FLI1low population, only the combination led to a significant increase in pH2AX. Furthermore, only the combination treatment led to a significant increase in pH2AX in the cancer stem cell population. Conclusions: We have identified that the combination of romidepsin with IE, but not IE alone, leads to an increase in DNA damage in the EWS:FLI1low and cancer stem cell populations. This suggests that the addition of romidepsin to SOC may improve treatment efficacy against subpopulations of tumor cells that are known to be chemoresistant. Further, this study highlights the use of CyTOF to assess treatment effects within ES tumor subpopulations to identify therapies that may be more effective against chemoresistant populations. Citation Format: Kaitlyn H. Smith, Lucy Endean, Kimberly Q. McKinney, Poornima Gourabathini, Kenzie Wells, Jeffrey Huo, Erin M. Trovillion, Javier Oesterheld, . CyTOF analysis of targeted therapy in addition to standard of care against Ewing sarcoma tumor subpopulations abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5695.