Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that predominantly affects children and adolescents. CD99 is a cell surface protein that is highly expressed on ES cells and is required to maintain their malignancy. We screened small molecule libraries for binding to extracellular domain of recombinant CD99 and subsequent inhibition of ES cell growth. We identified two structurally similar FDA-approved compounds, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in culture, suggesting that it can function through inhibiting CD99 independent of DNA metabolism. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting growth of three different ES xenografts. Our findings provide a novel molecular mechanism for clofarabine that involves direct binding to a cell surface receptor CD99 and inhibiting its biological activities.
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We thank the NCI/DPT Open Chemical Repository for providing compounds. We thank Dr. Abraham T. Kallarakal for help with the SPR screening experiments, Kelli Schanze for assistance with the animal experiments and Giulia Ricci for technical assistance. We thank Dr. Geeta Upadhyay for providing us the purified Ly6k protein. The authors thank Dr. Chand Khanna from the NCI/NIH (Bethesda, MD) for OS cell lines (HOS-MNNG and MG63.3), Dr. Eugenie S. Kleinerman from the University of Texas MD Anderson Cancer Center (Houston, TX) for human OS cell lines (SAOS-2 and SAOS-2/LM7), Dr. David M. Loeb from the Johns Hopkins university (Baltimore, MD) for MHH-ES cells, Dr. Heinrich Kovar from the Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung (Vienna, Austria) for STA-ET-7.2 cells, Dr. Timothy J. Triche from the Children’s Hospital (Los Angeles, CA) for 6647 cells, Dr. Angelo Rosolen from the University of Padova (Italy) and Dr. David N. Shapiro from the St. Jude Children’s Hospital (Memphis, TN) for rhabdomyosarcoma cell lines RH1, RH30 and RH4.
This work was supported by the funds from the Children's Cancer Foundation (to A. Üren), Hyundai Hope on Wheels (to A. Üren), the Alan B. Slifka Foundation (to A. Üren and K. Scotlandi) and the Italian Association for Cancer Research (AIRC_IG14049 to K. Scotlandi). The authors thank the Animal Models Shared Resource, Tissue Culture Shared Resource, Histopathology and Tissue Shared Resource and the Biacore Molecular Interaction Shared Resource at the Lombardi Comprehensive Cancer Center (Georgetown University), which are supported by a grant P30CA51008 from the National Cancer Institute.
Conflict of interest
Georgetown University has filed a patent application for the use of CD99 inhibitors in the treatment of Ewing sarcoma, in which Drs. AÜ, HÇ, and JAT was listed as inventors.
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Çelik, H., Sciandra, M., Flashner, B. et al. Clofarabine inhibits Ewing sarcoma growth through a novel molecular mechanism involving direct binding to CD99. Oncogene 37, 2181–2196 (2018). https://doi.org/10.1038/s41388-017-0080-4
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