American Association for Cancer Research
10780432ccr140492-sup-127353_2_supp_2665231_nc3b6g.doc (792.5 kB)

Supplementary Tables 1-6 from CD99 Triggering in Ewing Sarcoma Delivers a Lethal Signal through p53 Pathway Reactivation and Cooperates with Doxorubicin

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posted on 2023-03-31, 18:13 authored by Clara Guerzoni, Valentina Fiori, Mario Terracciano, Maria Cristina Manara, Diego Moricoli, Michela Pasello, Marika Sciandra, Giordano Nicoletti, Mara Gellini, Sabrina Dominici, Claudia Chiodoni, Pier Maria Fornasari, Pier-Luigi Lollini, Mario P. Colombo, Piero Picci, Maurizio Cianfriglia, Mauro Magnani, Katia Scotlandi

Supplementary Table S1: Efficacy of anti-CD99 mAb 0662 in a panel of EWS cell lines Supplementary Table S2: Fold induction/reduction of gene expression in treated samples over control mRNAs, analyzed with F-statistic (error <5%). Supplementary Table S3: Fold induction/reduction of protein expression in 5 min treated samples over control Supplementary Table S4: Ratio of firefly and Renilla luciferase signals in Saos-2 p53 deficient cell line co-transfected with a p53-reporter plasmid (pG13-LUC) and pCDNA3.1-E.V., pcDNA3.1-p53WT or mutant pcDNA3.1- p53S241F. Supplementary Table S5: Evaluation of NK number and activity in the four mice groups. Supplementary Table S6: Mice weight and blood parameters of mice from control or treated groups.



Purpose: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application.Experimental Design:In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7.Results: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS.Conclusions: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death. Clin Cancer Res; 21(1); 146–56. ©2014 AACR.