Clofarabine inhibits Ewing sarcoma growth through a novel molecular mechanism involving direct binding to CD99


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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  1. 1.

    Kovar H. Ewing’s sarcoma and peripheral primitive neuroectodermal tumors after their genetic union. Curr Opin Oncol. 1998;10:334–42.

    CAS  Article  Google Scholar 

  2. 2.

    Arndt CA, Crist WM. Common musculoskeletal tumors of childhood and adolescence. N Engl J Med. 1999;341:342–52.

    CAS  Article  Google Scholar 

  3. 3.

    Kovar H, Amatruda J, Brunet E, Burdach S, Cidre-Aranaz F, de Alava E, et al. The second European interdisciplinary Ewing sarcoma research summit—a joint effort to deconstructing the multiple layers of a complex disease. Oncotarget. 2016;7:8613–24.

    Article  Google Scholar 

  4. 4.

    Lawlor ER, Sorensen PH. Twenty years on: What do we really know about ewing sarcoma and what is the path forward? Crit Rev Oncog. 2015;20:155–71.

    Article  Google Scholar 

  5. 5.

    Gaspar N, Hawkins DS, Dirksen U, Lewis IJ, Ferrari S, Le Deley MC, et al. Ewing sarcoma: Current management and future approaches through collaboration. J Clin Oncol. 2015;33:3036–46.

    CAS  Article  Google Scholar 

  6. 6.

    Longhi A, Ferrari S, Tamburini A, Luksch R, Fagioli F, Bacci G, et al. Late effects of chemotherapy and radiotherapy in osteosarcoma and Ewing sarcoma patients: the Italian Sarcoma Group Experience (1983–2006). Cancer. 2012;118:5050–9.

    Article  Google Scholar 

  7. 7.

    Kovar H, Dworzak M, Strehl S, Schnell E, Ambros IM, Ambros PF, et al. Overexpression of the pseudoautosomal gene MIC2 in Ewing’s sarcoma and peripheral primitive neuroectodermal tumor. Oncogene. 1990;5:1067–70.

    CAS  PubMed  Google Scholar 

  8. 8.

    Ambros IM, Ambros PF, Strehl S, Kovar H, Gadner H, Salzer-Kuntschik M. MIC2 is a specific marker for Ewing’s sarcoma and peripheral primitive neuroectodermal tumors. Evidence for a common histogenesis of Ewing’s sarcoma and peripheral primitive neuroectodermal tumors from MIC2 expression and specific chromosome aberration. Cancer. 1991;67:1886–93.

    CAS  Article  Google Scholar 

  9. 9.

    Fellinger EJ, Garin-Chesa P, Triche TJ, Huvos AG, Rettig WJ. Immunohistochemical analysis of Ewing’s sarcoma cell surface antigen p30/32MIC2. Am J Pathol. 1991;139:317–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Perlman EJ, Dickman PS, Askin FB, Grier HE, Miser JS, Link MP. Ewing’s sarcoma—routine diagnostic utilization of MIC2 analysis: a Pediatric Oncology Group/Children’s Cancer Group Intergroup Study. Hum Pathol. 1994;25:304–7.

    CAS  Article  Google Scholar 

  11. 11.

    Weidner N, Tjoe J. Immunohistochemical profile of monoclonal antibody O13: antibody that recognizes glycoprotein p30/32MIC2 and is useful in diagnosing Ewing’s sarcoma and peripheral neuroepithelioma. Am J Surg Pathol. 1994;18:486–94.

    CAS  Article  Google Scholar 

  12. 12.

    Lee CS, Southey MC, Waters K, Kannourakis G, Georgiou T, Armes JE, et al. EWS/FLI-1 fusion transcript detection and MIC2 immunohistochemical staining in the diagnosis of Ewing’s sarcoma. Pediatr Pathol Lab Med 1996;16:379–92.

    CAS  Article  Google Scholar 

  13. 13.

    Halliday BE, Slagel DD, Elsheikh TE, Silverman JF. Diagnostic utility of MIC-2 immunocytochemical staining in the differential diagnosis of small blue cell tumors. Diagn Cytopathol. 1998;19:410–6.

    CAS  Article  Google Scholar 

  14. 14.

    Lucas DR, Bentley G, Dan ME, Tabaczka P, Poulik JM, Mott MP. Ewing sarcoma vs lymphoblastic lymphoma. A comparative immunohistochemical study. Am J Clin Pathol. 2001;115:11–17.

    CAS  Article  Google Scholar 

  15. 15.

    Khoury JD. Ewing sarcoma family of tumors. Adv Anat Pathol. 2005;12:212–20.

    Article  Google Scholar 

  16. 16.

    Aubrit F, Gelin C, Pham D, Raynal B, Bernard A. The biochemical characterization of E2, a T cell surface molecule involved in rosettes. Eur J Immunol. 1989;19:1431–6.

    CAS  Article  Google Scholar 

  17. 17.

    Gelin C, Aubrit F, Phalipon A, Raynal B, Cole S, Kaczorek M, et al. The E2 antigen, a 32 kd glycoprotein involved in T-cell adhesion processes, is the MIC2 gene product. EMBO J. 1989;8:3253–9.

    CAS  Article  Google Scholar 

  18. 18.

    Ellis NA, Tippett P, Petty A, Reid M, Weller PA, Ye TZ, et al. PBDX is the XG blood group gene. Nat Genet. 1994;8:285–90.

    CAS  Article  Google Scholar 

  19. 19.

    Suh YH, Shin YK, Kook MC, Oh KI, Park WS, Kim SH, et al. Cloning, genomic organization, alternative transcripts and expression analysis of CD99L2, a novel paralog of human CD99, and identification of evolutionary conserved motifs. Gene. 2003;307:63–76.

    CAS  Article  Google Scholar 

  20. 20.

    Bixel G, Kloep S, Butz S, Petri B, Engelhardt B, Vestweber D. Mouse CD99 participates in T-cell recruitment into inflamed skin. Blood. 2004;104:3205–13.

    CAS  Article  Google Scholar 

  21. 21.

    Dworzak MN, Fritsch G, Fleischer C, Printz D, Froschl G, Buchinger P, et al. CD99 (MIC2) expression in paediatric B-lineage leukaemia/lymphoma reflects maturation-associated patterns of normal B-lymphopoiesis. Br J Haematol. 1999;105:690–5.

    CAS  Article  Google Scholar 

  22. 22.

    Schenkel AR, Mamdouh Z, Chen X, Liebman RM, Muller WA. CD99 plays a major role in the migration of monocytes through endothelial junctions. Nat Immunol. 2002;3:143–50.

    CAS  Article  Google Scholar 

  23. 23.

    Hu-Lieskovan S, Zhang J, Wu L, Shimada H, Schofield DE, Triche TJ. EWS-FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing’s family of tumors. Cancer Res. 2005;65:4633–44.

    CAS  Article  Google Scholar 

  24. 24.

    Miyagawa Y, Okita H, Nakaijima H, Horiuchi Y, Sato B, Taguchi T, et al. Inducible expression of chimeric EWS/ETS proteins confers Ewing’s family tumor-like phenotypes to human mesenchymal progenitor cells. Mol Cell Biol. 2008;28:2125–37.

    CAS  Article  Google Scholar 

  25. 25.

    Rocchi A, Manara MC, Sciandra M, Zambelli D, Nardi F, Nicoletti G, et al. CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest. 2010;120:668–80.

    CAS  Article  Google Scholar 

  26. 26.

    Franzetti GA, Laud-Duval K, Bellanger D, Stern MH, Sastre-Garau X, Delattre O. MiR-30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor. Oncogene. 2013;32:3915–21.

    CAS  Article  Google Scholar 

  27. 27.

    Ventura S, Aryee DN, Felicetti F, De Feo A, Mancarella C, Manara MC, et al. CD99 regulates neural differentiation of Ewing sarcoma cells through miR-34a-Notch-mediated control of NF-kappaB signaling. Oncogene. 2016;35:3944–54.

    CAS  Article  Google Scholar 

  28. 28.

    Sohn HW, Choi EY, Kim SH, Lee IS, Chung DH, Sung UA, et al. Engagement of CD99 induces apoptosis through a calcineurin-independent pathway in Ewing’s sarcoma cells. Am J Pathol. 1998;153:1937–45.

    CAS  Article  Google Scholar 

  29. 29.

    Scotlandi K, Baldini N, Cerisano V, Manara MC, Benini S, Serra M, et al. CD99 engagement: an effective therapeutic strategy for Ewing tumors. Cancer Res. 2000;60:5134–42.

    CAS  PubMed  Google Scholar 

  30. 30.

    Kreppel M, Aryee DN, Schaefer KL, Amann G, Kofler R, Poremba C, et al. Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing’s sarcoma cells. Oncogene. 2006;25:2795–2800.

    CAS  Article  Google Scholar 

  31. 31.

    Scotlandi K, Perdichizzi S, Bernard G, Nicoletti G, Nanni P, Lollini PL, et al. Targeting CD99 in association with doxorubicin: an effective combined treatment for Ewing’s sarcoma. Eur J Cancer. 2006;42:91–6.

    CAS  Article  Google Scholar 

  32. 32.

    Manara MC, Bernard G, Lollini PL, Nanni P, Zuntini M, Landuzzi L, et al CD99 acts as an oncosuppressor in osteosarcoma. Mol Biol Cell. 2006;17:1910–21.

    CAS  Article  Google Scholar 

  33. 33.

    Zucchini C, Manara MC, Pinca RS, De Sanctis P, Guerzoni C, Sciandra M, et al. CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity. Oncogene. 2014;33:1912–21.

    CAS  Article  Google Scholar 

  34. 34.

    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–54.

    CAS  Article  Google Scholar 

  35. 35.

    Brohl AS, Solomon DA, Chang W, Wang J, Song Y, Sindiri S, et al. The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation. PLoS Genet. 2014;10:e1004475.

    Article  Google Scholar 

  36. 36.

    Choi G, Lee SW, Jung KC, Choi EY. Detection of homodimer formation of CD99 through extracelluar domain using bimolecular fluorescence complementation analysis. Exp Mol Med. 2007;39:746–55.

    CAS  Article  Google Scholar 

  37. 37.

    Lee MK, Kim HS, Kim SS, Cho MH, Lee IS. Analysis of the dimerization of human CD99 using bimolecular fluorescence complementation technique. J Microbiol Biotechnol. 2008;18:472–6.

    CAS  PubMed  Google Scholar 

  38. 38.

    Kim HJ, Chong KH, Kang SW, Lee JR, Kim JY, Hahn MJ, et al. Identification of cyclophilin A as a CD99-binding protein by yeast two-hybrid screening. Immunol Lett. 2004;95:155–9.

    CAS  Article  Google Scholar 

  39. 39.

    Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, et al. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration. J Exp Med. 2015;212:1021–41.

    CAS  Article  Google Scholar 

  40. 40.

    Gollnest T, de Oliveira TD, Schols D, Balzarini J, Meier C. Lipophilic prodrugs of nucleoside triphosphates as biochemical probes and potential antivirals. Nat Commun. 2015;6:8716.

    CAS  Article  Google Scholar 

  41. 41.

    Gollnest T, Dinis de Oliveira T, Rath A, Hauber I, Schols D, Balzarini J, et al. Membrane-permeable triphosphate prodrugs of nucleoside analogues. Angew Chem. 2016;55:5255–8.

    CAS  Article  Google Scholar 

  42. 42.

    Gellini M, Ascione A, Flego M, Mallano A, Dupuis ML, Zamboni S, et al. Generation of human single-chain antibody to the CD99 cell surface determinant specifically recognizing Ewing’s sarcoma tumor cells. Curr Pharm Biotechnol. 2013;14:449–63.

    CAS  Article  Google Scholar 

  43. 43.

    Guerzoni C, Fiori V, Terracciano M, Manara MC, Moricoli D, Pasello M, et al. CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin. Clin Cancer Res. 2015;21:146–56.

    CAS  Article  Google Scholar 

  44. 44.

    Moricoli D, Carbonella DC, Dominici S, Fiori V, Balducci MC, Guerzoni C, et al. Process development of a human recombinant diabody expressed in E. coli: engagement of CD99-induced apoptosis for target therapy in Ewing’s sarcoma. Appl Microbiol Biotechnol. 2016;100:3949–63.

    CAS  Article  Google Scholar 

  45. 45.

    Bonate PL, Arthaud L, Cantrell WR Jr., Stephenson K, Secrist JA 3rd, Weitman S. Discovery and development of clofarabine: a nucleoside analogue for treating cancer. Nat Rev Drug Discov. 2006;5:855–63.

    CAS  Article  Google Scholar 

  46. 46.

    Sigal DS, Miller HJ, Schram ED, Saven A. Beyond hairy cell: the activity of cladribine in other hematologic malignancies. Blood. 2010;116:2884–96.

    CAS  Article  Google Scholar 

  47. 47.

    Kawasaki H, Carrera CJ, Piro LD, Saven A, Kipps TJ, Carson DA. Relationship of deoxycytidine kinase and cytoplasmic 5’-nucleotidase to the chemotherapeutic efficacy of 2-chlorodeoxyadenosine. Blood. 1993;81:597–601.

    CAS  PubMed  Google Scholar 

  48. 48.

    Chung SS, Eng WS, Hu W, Khalaj M, Garrett-Bakelman FE, Tavakkoli M, et al. CD99 is a therapeutic target on disease stem cells in myeloid malignancies. Sci Transl Med. 2017;9:eaaj2025.

    Article  Google Scholar 

  49. 49.

    Bernard A, Aubrit F, Raynal B, Pham D, Boumsell LA. T cell surface molecule different from CD2 is involved in spontaneous rosette formation with erythrocytes. J Immunol. 1988;140:1802–7.

    CAS  PubMed  Google Scholar 

  50. 50.

    Dworzak MN, Fritsch G, Buchinger P, Fleischer C, Printz D, Zellner A, et al. Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood. Blood. 1994;83:415–25.

    CAS  PubMed  Google Scholar 

  51. 51.

    Husak Z, Printz D, Schumich A, Potschger U, Dworzak MN. Death induction by CD99 ligation in TEL/AML1-positive acute lymphoblastic leukemia and normal B cell precursors. J Leukoc Biol. 2010;88:405–12.

    CAS  Article  Google Scholar 

  52. 52.

    Husak Z, Dworzak MN. CD99 ligation upregulates HSP70 on acute lymphoblastic leukemia cells and concomitantly increases NK cytotoxicity. Cell death Dis. 2012;3:e425.

    CAS  Article  Google Scholar 

  53. 53.

    Cox CV, Diamanti P, Moppett JP, Blair A. Investigating CD99 expression in leukemia propagating cells in childhood T cell acute lymphoblastic leukemia. PLoS One. 2016;11:e0165210.

    Article  Google Scholar 

  54. 54.

    Chung SS, Tavakkoli M, Devlin SM, Park CY. CD99 Is a therapeutic target on disease stem cells in acute myeloid leukemia and the myelodysplastic syndromes. Blood. 2013;122:2891.

    Article  Google Scholar 

  55. 55.

    Chung SS, Tavakkoli M, Klimek VM, Park CY. CD99 is a therapeutic target on disease initiating stem cells in acute myeloid leukemia and the myelodysplastic syndromes. Blood. 2014;124:3760.

    Google Scholar 

  56. 56.

    Winger RC, Harp CT, Chiang MY, Sullivan DP, Watson RL, Weber EW, et al. Cutting edge: CD99 is a novel therapeutic target for control of T cell-mediated central nervous system autoimmune disease. J Immunol. 2016;196:1443–8.

    CAS  Article  Google Scholar 

  57. 57.

    Giovannoni G, Comi G, Cook S, Rammohan K, Rieckmann P, Soelberg Sorensen P, et al. A placebo-controlled trial of oral cladribine for relapsing multiple sclerosis. N Engl J Med. 2010;362:416–26.

    CAS  Article  Google Scholar 

  58. 58.

    Celik H, Sajwan KP, Selvanathan SP, Marsh BJ, Pai AV, Kont YS, et al. Ezrin binds to DEAD-box RNA helicase DDX3 and regulates its function and protein level. Mol Cell Biol. 2015;35:3145–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.

    CAS  Article  Google Scholar 

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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.

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Correspondence to Katia Scotlandi or Aykut Üren.

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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).

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