Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

Increased avidity and efficacy of dAbd over scFv C7 in 6647 EWS cell line.

Effects of 0662 on Gadd45-α mRNA expression and osteoblastic differentiation on mesenchymal stem cells.

Effects of combined treatment with doxorubicin and dAbd C7.

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.

p53 is necessary for CD99-induced cell-death and is phosphorilated by 0662.

Supplementary Methods from CD99 Triggering in Ewing Sarcoma Delivers a Lethal Signal through p53 Pathway Reactivation and Cooperates with Doxorubicin

Supplementary Data from Prognostic Value of CCN3 in Osteosarcoma

HSP70 levels are unaffected from dAbd C7 treatment.

dAbd C7 activity is related to p53 status, but is ineffective in h-MSC cells.

Appropriate tools for monitoring sarcoma progression are still limited. The aim of the present study was to investigate the value of miR-34a-5p (miR34a) as a circulating biomarker to follow disease progression and measure the therapeutic response. Stable forced re-expression of miR34a in Ewing sarcoma (EWS) cells significantly limited tumor growth in mice. Absolute quantification of miR34a in the plasma of mice and 31 patients showed that high levels of this miRNA inversely correlated with tumor volume. In addition, miR34a expression was higher in the blood of localized EWS patients than in the blood of metastatic EWS patients. In 12 patients, we followed miR34a expression during preoperative chemotherapy. While there was no variation in the blood miR34a levels in metastatic patients at the time of diagnosis or after the last cycle of preoperative chemotherapy, there was an increase in the circulating miR34a levels in patients with localized tumors. The three patients with the highest fold-increase in the miR levels did not show evidence of metastasis. Although this analysis should be extended to a larger cohort of patients, these findings imply that detection of the miR34a levels in the blood of EWS patients may assist with the clinical management of EWS.

Ewing sarcoma (EWS) is an aggressive mesenchymal tumor with unmet clinical need and significant social impacts on children, adolescents, and young adults. CD99, a hallmark surface molecule of EWS, participates in crucial biological processes including cell migration, differentiation, and death. EWS cells can release CD99 through exosomes (EXOs), specialized extracellular vesicles with major cell communication roles. Here we show that, as a consequence of CD99 silencing, EWS cells deliver exosomes with oncosuppressive functions that significantly reduce tumor aggressiveness. These CD99-lacking microvesicles modulate gene expression of the EWS-recipient cells, reduce proliferation and migration, in turn inducing a more-differentiated less-malignant phenotype. The most relevant effects were detected on the activator protein-1 signaling pathway whose regulation was found to be dependent on the specific cargo loaded in vesicles after CD99 shutdown. Investigation of the miRNA content of CD99-deprived EXOs identified miR-199a-3p as a key driver able to reverse EWS malignancy in experimental models as well as in clinical specimens. All together, our data provide evidence that the abrogation of CD99 in EWS tumor cells leads to produce and release EXOs capable to transfer their antineoplastic effects into the nearby tumor cells, suggesting a novel atypical role for these microvesicles in reversion of malignancy rather than in priming the soil for progression and metastatic seeding. This conceptually innovative approach might offer a new therapeutic opportunity to treat a tumor still refractory to most treatments.

Abstract Background Ewing sarcoma (EWS) is an aggressive childhood bone tumor with still highly unmet clinical needs. The tumor is characterized by the fusion oncoprotein EWS-FLI1 and the high expression of the membrane glycoprotein CD99. CD99 has been found to be released by EWS cells linked to exosomes (EXOs), small vesicles able to influence cellular behavior and surrounding microenvironment by transferring their contents into nearby or distant recipient cells. The delivery of EXOs devoid of CD99 was sufficient to induce neural differentiation in recipient EWS cells through miR- 34a-dependent inhibition of Notch-NF-kB signaling, thus indicating a possible therapeutic potential. Aims The study has the ambition to: 1. Evaluate the capabilities of EXOs released by Ewing's sarcoma, to influence cancer growth and/or metastasis through the horizontal transfer of their cargos. 2. Characterize EXOs secreted by CD99-silenced EWS cells in terms of miRNAs and protein and evaluate their general antimetastatic function, if any, taking advantage of their capabilities to be transferred into a broad panel of different Ewing's sarcoma cell lines, as recipients. Results In the present study we identified the role of Ewing sarcoma-derived EXOs as mediators of signals involved in cancer growth, metastases and differentiation. In particular, we analyzed the ability of EXOs, expressing or not CD99, to modulate the phenotype of EWS cells. Delivery of EXOs from CD99-silenced cells was sufficient to induce neural differentiation in recipient EWS cells by neurite outgrowth and increased expression of β-III tubulin marker of neural differentiation accompanied in vitro by a significant reduction of proliferation, invasion and chemotaxis convey the same phenotype obtained by stable CD99 silencing. Moreover, microarray analysis revealed a signature of 56 miRNAs differentially expressed in EXOs derived from CD99-silenced cells compared to EWS-derived EXOs. We focused our attention on miR-214 and miR-199. Those miRNAs are involved in the regulation of CD99 itself like, EWS-FLI1 or CD99-mediated pathways related to cytoskeleton and membrane organization. As a next step we searched for any tumorigenic effect possibly associated with miR-199 and mir-214. The restored expression of miR-199 and miR-214 in two Ewing Sarcoma cell lines was accompanied by a significant reduction of proliferation, invasion and chemotaxis in vitro. The outcomes deriving from miR-199 and miR-214 enforced expression were also evaluated on capability of forming foci in agar semisolid medium. Results showed a significant decrease of the number of foci in both cell lines. Conclusions Exosomes deprived of CD99 can alter the balance of important cellular components necessary for the metastatic process of EWS cells, thus representing a novel exciting therapeutic possibility for the design of novel therapy against metastatic EWS. AIRC IG18451 to KS; IG18815 to AC. Citation Format: Alessandra De Feo, Marika Sciandra, Federica Felicetti, Manuela Ferracin, Alessandra Carè, Katia Scotlandi. Exosome-mediated transfer of sh-CD99 is sufficient to modulate cell differentiation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3549.

Abstract Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy of unknown primary origin. ES cells express high levels of CD99, a cell surface protein routinely used as a marker antigen in the histologic diagnosis of ES. A substantial body of evidence makes CD99 an attractive therapeutic target for patients with ES. We identified two structurally similar FDA-approved nucleoside (purine) analogues, clofarabine and cladribine, as specific CD99 inhibitors in a Biacore screening experiment. In validation experiments, both drugs exhibited selective cytotoxicity toward ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A membrane-impermeable derivative of clofarabine, clofarabine-5′-triphosphate, showed a similar cytotoxicity on ES cell lines, suggesting inhibition of CD99 activity on cell surface as a likely mechanism by which the drug exerts its cytotoxic action without any effect on DNA metabolism. Clofrabine also inhibited the growth of three different ES xenografts in vivo. We performed a phosphokinase array to identify the mechanism behind how clofarabine may regulate cellular functions through inhibiting CD99. Mitogen- and stress-activated kinases 1 and 2 (MSK1/2) were the most significantly activated proteins by both CD99 antibody and clofarabine. Four of the 7 proteins with increased phosphorylation were related to MSK1/2, which were MSK1/2 itself, its substrates CREB and c-Jun, and its upstream kinase ERK1/2, thus representing the most likely intracellular pathway responsible for cell death due to CD99 inhibition. Clofarabine induced a significant increase in phosphorylation levels of MSK1/2 in cohort of ES xenografts, supporting its use as a potential pharmacodynamic marker of CD99 inhibition. We compared the activity of clofarabine with cytarabine, a structurally similar pyrimidine analog that has been identified as an inhibitor of EWS/FLI1 transcriptional activity but failed in clinical trials on ES patients. Cytarabine did not activate MSK1/2 phosphorylation in ES cells, suggesting that clofarabine may function through alternative mechanisms on ES cells that are different than cytarabine. Overall, our findings suggest that clofarabine directly binds to CD99 and inhibits its oncogenic activity through a novel mechanism in ES, and therefore it is a good candidate for early-phase clinical trials in children with ES. Citation Format: Haydar Çelik, Marika Sciandra, Maria Cristina Manara, Jeffrey Torestky, Katia Scotlandi, Aykut Üren. Inhibition of CD99 activity by clofarabine as a novel therapeutic for Ewing sarcoma involves a novel molecular mechanism that is different than cytarabine [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A06.

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.

Abstract Ewing sarcoma (ES) is an aggressive bone and soft tissue malignancy that affects predominantly children and adolescents with a high propensity to metastasize and poor prognosis. CD99 is a transmembrane cell surface protein that is highly expressed on ES cells, and routinely used as a marker for histological diagnosis of ES. We screened small molecule libraries for their binding to recombinant CD99 protein and subsequent selective inhibition of ES cell growth. We identified two structurally similar FDA-approved nucleoside analogues, clofarabine and cladribine that selectively inhibited the growth of ES cells in a panel of 14 ES vs. 28 non-ES cell lines. A significant negative correlation was found in human cell lines between CD99 expression and IC50 values for clofarabine and cladribine. Both drugs inhibited CD99 dimerization and its interaction with downstream signaling components cyclophilin A and PKA-RIIα as well as led to reduced ROCK2 protein expression and migration in ES cells. A membrane-impermeable analog of clofarabine showed similar cytotoxicity in ES cells, suggesting that it can function through inhibiting CD99 alone without any effect on DNA metabolism. Clofarabine and cladribine led to a significant increase in hypodiploid DNA content of ES cells, which was diminished by suppression of CD99 expression. Both drugs drastically inhibited anchorage-independent growth of ES cells, but clofarabine was more effective in inhibiting ES xenografts. Finally, the screening of a set of chemotherapy drugs revealed a synergy for the combination of anti-CD99 drugs and dasatinib in ES cells, which may translate into increased survival and reduced toxicity. Overall, our findings suggest that clofarabine is a good candidate for early phase clinical trials in children with ES. Citation Format: Haydar Celik, Marika Sciandra, Bess Flashner, Elif Gelmez, Neslihan Kayraklıoğlu, David V. Allegakoen, Jeff R. Petro, Erin J. Conn, Sarah Hour, Jenny Han, Lalehan Oktay, Purushottam B. Tiwari, Mutlu Hayran, Maria Cristina Manara, Jeffrey A. Toretsky, Katia Scotlandi, Aykut Uren. Discovery of first-in-class small molecule CD99 inhibitors for targeted therapy of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1933. doi:10.1158/1538-7445.AM2017-1933

CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

Background: microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate.Methods: Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed.Results: We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs.Conclusions: These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.

AbstractPurpose: 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.