CD99 Expression and Prognostic Impact in Glioblastoma: A Single-Center Cohort Study.

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan-Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma.

CD99 Modulates the Proteomic Landscape of Ewing Sarcoma Cells and Related Extracellular Vesicles.

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.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

Childhood pilomatrixoma mimicking malignant small round blue cell tumor with positivity for CD99: Potential pitfall in cytology.

Pilomatrixoma is a relatively rare benign skin appendageal tumor, often presenting in the pediatric age group as a nodular lesion and most commonly involving the head and neck, making it amenable to primary fine needle aspiration (FNA) diagnosis. We report the clinical and histopathological findings of two cases of pilomatrixoma in children, both of which were initially misdiagnosed as small round blue cell tumors due to high cellularity and misinterpretation of the proliferating basaloid cells. Histopathology revealed basal cell proliferation and mitoses indicating that they were progressive, early lesions. The first case showed membranous positivity for CD99 which prompted a diagnosis of Ewing sarcoma. Awareness of the morphological spectrum including positivity for CD99 and careful evaluation of cell block histology could have averted the misdiagnosis. Pilomatrixoma should be included as an important differential diagnosis when faced with primitive-appearing cells on FNA, especially in children with mass lesions in the head and neck region.

CD99 and NKX2.2 positive neuroblastoma diagnosed on cytology: A potential diagnostic pitfall and necessity of pathological evaluation of the primary site.

Immunohistochemistry plays a vital role in the diagnosis of small round cell tumors. CD99 immunonegativity is one of the features, which helps in distinguishing neuroblastoma from other small round cell tumors. NKX2.2 is a specific marker of Ewing sarcoma, which is a differential for poorly differentiated neuroblastoma. Here, we present a case of metastatic neuroblastoma showing immunoreactivity for both CD99 and NKX2.2 on cytology of the metastatic site causing diagnostic dilemma. Biopsy study of the adrenal lesion revealed presence of differentiating cells and neuropil, highlighting the importance of evaluation of the primary site and limitation of cytology.

[MIC2 (CD99) as a diagnostic marker for TdT-negative B lymphoblastic lymphoma].

Lymphoblastic lymphoma (LBL) is a rare hematologic malignancy that originates from immature lymphocytes and usually expresses terminal deoxynucleotidyl transferase (TdT). Here, we report a case of TdT-negative B-LBL. A 71-year-old male patient presented to a hospital with shortness of breath. His chest computed tomography showed a mediastinal mass. Tumor cells did not express TdT but expressed MIC2, which led to LBL diagnosis. MIC2 is a useful marker for LBL diagnosis.

Design, synthesis and biological evaluation of Nucleosidic CD99 inhibitors that selectively reduce Ewing sarcoma viability.

Ewing Sarcoma (ES) is a cancer of bone and soft tissues affecting mostly children and young adults. Aggressive progression and poor prognosis of this malignancy call for novel and targeted treatments. CD99 is a transmembrane protein that is abundantly expressed on ES cells and is a diagnostic marker for the disease. ES cells are selectively sensitive to CD99 inhibition compared to most normal cells and other tumors. Therefore, CD99 is a good molecular target for ES treatment. Clofarabine and cladribine are two FDA approved drugs that are administered for their inhibitory acts on DNA synthesis to treat relapsed or refractory acute lymphoblastic and myeloid leukemia. They have also been shown to directly bind to CD99 and inhibit ES growth through a distinct mechanism. In the current study, we designed, synthesized and tested new ES specific derivatives of both drugs that would continue to target CD99 but with expected reduction in cellular membrane permeability and rendered unsuitable for inhibiting DNA synthesis. By using commercially available clofarabine and cladribine purine nucleoside analogs, we modified the primary alcohol moiety at the deoxyribose C-5' terminal site to suppress phosphorylation and thus inhibition of subsequent DNA synthesis pathways. In addition, we incorporated a variety of polar groups in the ribose and purine rings to reduce membrane permeability and investigated the effects of configurational changes in the sugar moiety. Among 26 new derivatives, we identified two compounds, BK50164 and BK60106, that cause cell death specifically in ES primarily due to inhibition of CD99 but not via inhibition of DNA synthesis. These findings provide a road map for the future development selective CD99 inhibitors for targeted treatment of ES.

Development and application of an indirect ELISA for the serological detection of bovine viral diarrhea virus infection based on the protein E2 antigen.

BACKGROUND: Bovine viral diarrhea virus (BVDV) causes continuous economic losses to the livestock industry. Monitoring antibodies with enzyme-linked immunosorbent assay (ELISA) is a valuable tool to ensure the purification of BVDV in cattle. However, currently available ELISA kits based on the whole BVDV virion are both costly and time-consuming. The E2 protein has good immunogenicity, induces the secretion of neutralizing antibodies and is an essential immunogen for serological detection. METHODS AND RESULTS: We developed a novel recombinant E2 protein-based indirect ELISA (rE2-iELISA) and conducted a serological survey for BVDV antibodies in 2021-2022 in Beijing, China. The results showed that E2 protein was successfully expressed with high immunogenicity and the optimal rE2-iELISA displayed high sensitivity, reproducibility and specificity. Clinical testing of 566 serum specimens indicated that 318 BVDV positive samples and 194 BVDV negative samples were tested by rE2-iELISA and the IDEXX BVDV ELISA-Ab kit, with a positive coincidence rate of 93.3%, a negative coincidence rate of 86.3%, and an overall coincidence rate of 90.5%. CONCLUSION: This study established an rE2-iELISA method, which is a highly sensitive, specific and robust ELISA-test validated to detect anti-BVDV antibodies. These findings indicate that the newly developed rE2-iELISA method has the potential to be used as a rapid, reliable and cost-effective screening tool for BVDV infection and provides technical support for the evaluation of vaccine efficacy in cattle herds in the future.

CD99 and the Chicken Alloantigen D Blood System.

The chicken D blood system is one of 13 alloantigen systems found on chicken red blood cells. Classical recombinant studies located the D blood system on chicken chromosome 1, but the candidate gene was unknown. Multiple resources were utilized to identify the chicken D system candidate gene, including genome sequence information from both research and elite egg production lines for which D system alloantigen alleles were reported, and DNA from both pedigree and non-pedigree samples with known D alleles. Genome-wide association analyses using a 600 K or a 54 K SNP chip plus DNA from independent samples identified a strong peak on chicken chromosome 1 at 125-131 Mb (GRCg6a). Cell surface expression and the presence of exonic non-synonymous SNP were used to identify the candidate gene. The chicken CD99 gene showed the co-segregation of SNP-defined haplotypes and serologically defined D blood system alleles. The CD99 protein mediates multiple cellular processes including leukocyte migration, T-cell adhesion, and transmembrane protein transport, affecting peripheral immune responses. The corresponding human gene is found syntenic to the pseudoautosomal region 1 of human X and Y chromosomes. Phylogenetic analyses show that CD99 has a paralog, XG, that arose by duplication in the last common ancestor of the amniotes.

EREG is the core onco-immunological biomarker of cuproptosis and mediates the cross-talk between VEGF and CD99 signaling in glioblastoma.

BACKGROUND: Glioma is the most prevalent primary tumor of the central nervous system. Glioblastoma multiforme (GBM) is the most malignant form of glioma with an extremely poor prognosis. A novel, regulated cell death form of copper-induced cell death called "cuproptosis" provides a new prospect for cancer treatment by regulating cuproptosis. METHODS: Data from bulk RNA sequencing (RNA-seq) analysis (The Cancer Genome Atlas cohort and Chinese Glioma Genome Atlas cohort) and single cell RNA-seq (scRNA-seq) analysis were integrated to reveal their relationships. A scoring system was constructed according to the cuproptosis-related gene expression, and core genes were experimentally verified using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot (WB), immunohistochemistry (IHC), and immunofluorescence (IF). Moreover, cell counting kit-8 (CCK8), colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, transwell, and flow cytometry cell cycle were performed to evaluate cell proliferation, invasion, and migration. RESULTS: The Cuproptosis Activation Scoring (CuAS) Model has stable and independent prognostic efficacy, as verified by two CGGA datasets. Epiregulin (EREG), the gene of the model has the most contributions in the principal component analysis (PCA), is an onco-immunological gene that can affect immunity by influencing the expression of programmed death-ligand 1 (PD-L1) and mediate the process of cuproptosis by influencing the expression of ferredoxin 1 (FDX1). Single cell transcriptome analysis revealed that high CuAS GBM cells are found in vascular endothelial growth factor A (VEGFA) + malignant cells. Oligodendrocyte transcription factor 1 (OLIG1) + malignant is the original clone, and VEGF and CD99 are the differential pathways of specific cell communication between the high and low CuAS groups. This was also demonstrated by immunofluorescence in the tissue sections. Furthermore, CuAS has therapeutic potential for immunotherapy, and we predict that many drugs (methotrexate, NU7441, KU -0063794, GDC-0941, cabozantinib, and NVP-BEZ235) may be used in patients with high CuAS. CONCLUSION: EREG is the core onco-immunological biomarker of CuAS and modulates the cross-talk between VEGF and CD99 signaling in glioblastoma, and CuAS may provide support for immunotherapy and chemotherapy.

Correlation NKX2.2 IHC and EWSR1 break-apart FISH in the diagnosis of Ewing sarcoma: Can combined NKX2.2 and CD99 immunoexpression obviate or minimize the need of FISH testing? First assessment study from Indian tertiary cancer care center.

Context: Ewing sarcoma (ES) are malignant small round cell tumors (MSRCT) characterized by rearrangements of EWSR1 gene. Although gold standard for diagnosis is detection of specific fusion genes by molecular testing, these ancillary tests are costly and only available in limited number of settings. There is a persuasive evidence for reliability of NKX2.2 immunohistochemistry (IHC) as a surrogate marker for EWSR1 gene rearrangement in ES. Aims: The aim of this study is to correlate the NKX2.2 immuno-expression with genetically confirmed ES cases and also to assess the reliability and accuracy of NKX2.2 along with combined positivity of NXX2.2 and CD99 in diagnosing ES and differentiating it from other relevant histological mimics. Settings and Design: The present study is a retrospective study conducted over a period of 6-year duration in a tertiary cancer care center. Methods and Material: We evaluated NKX2.2 immunoexpression in 35 genetically confirmed cases of ES and also in pertaining differential entities (n = 58) of ES including rhabdomyosarcoma (n = 20), lymphoblastic lymphoma (n = 14), Wilms tumor (n = 10), poorly differentiated synovial sarcoma (n = 4), small-cell osteosarcoma (n = 4), neuroblastoma (n = 5), and mesenchymal chondrosarcoma (n = 1). CD99 was performed in the category of MSRCTs showing NKX2.2 positivity to evaluate combined specificity for the diagnosis of ES. Results: Of the 35 genetically confirmed cases of ES, 29 cases (83%) showed NKX2.2-positive expression (83% sensitivity). Compared to ES, NKX2.2 was positive in only 05% cases (3/58 cases) of non-ES MSRCT. Only two of five cases of neuroblastomas and one case of mesenchymal chondrosarcoma showed NKX2.2 positivity. CD99 positivity was seen in 100% of ES and in the single case of mesenchymal chondrosarcoma. All five cases (100%) of neuroblastoma were negative for CD99. Conclusions: The presented study, which is the first from an Indian oncology center, showed NKX2.2 IHC is quite reliable in diagnosis of ES in the right clinicopathological context. With remarkable sensitivity and specificity of NKX2.2 IHC for diagnosis of ES, we propose that combined positivity of CD99 and NKX2.2 IHC can obviate or minimize the need of EWSR1 gene rearrangement molecular testing for diagnosis of ES.

Cell Adhesion Molecule CD99 in Cancer Immunotherapy.

The CD99 antigen is a transmembrane protein expressed in a broad variety of tissues, particularly in hematopoietic cells, thymus, endothelial cells, etc. It participates in several crucial biological processes, including cell adhesion, migration, death, differentiation, and inflammation. CD99 has shown oncogenic or tumor suppressor roles in different types of cancer. Therefore, it has been used as a biomarker and therapeutic target for several types of cancer. Moreover, it has also been reported to be involved in several critical immune processes, such as T cell activation and differentiation, dendritic cell differentiation, and so on. Hence, CD99 may have potential values in cancer immunotherapy. Anti-CD99 antibodies have shown therapeutic effects on certain types of cancer, especially on Ewing sarcoma and T cell acute lymphoblastic leukemia (ALL). This review summarizes the recent progress of CD99 in cancer research and targeting therapies, especially in cancer immunotherapy, which may help researchers understand the crucial roles of CD99 in cancer development and design new therapeutic strategies.

Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells.

CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production.

Human CD99L2 Regulates a Unique Step in Leukocyte Transmigration.

CD99-like 2 (CD99L2 [L2]) is a highly glycosylated 52-kDa type 1 membrane protein that is important for leukocyte transendothelial migration (TEM) in mice. Inhibiting L2 using function-blocking Ab significantly reduces the recruitment of leukocytes to sites of inflammation in vivo. Similarly, L2 knockout mice have an inherent defect in leukocyte transmigration into sites of inflammation. However, the role of L2 in inflammation has only been studied in mice. Furthermore, the mechanism by which it regulates TEM is not known. To study the relevance to human inflammation, we studied the role of L2 on primary human cells in vitro. Our data show that like PECAM and CD99, human L2 is constitutively expressed at the borders of endothelial cells and on the surface of leukocytes. Inhibiting L2 using Ab blockade or genetic knockdown significantly reduces transmigration of human neutrophils and monocytes across endothelial cells. Furthermore, our data also show that L2 regulates a specific, sequential step of TEM between PECAM and CD99, rather than operating in parallel or redundantly with these molecules. Similar to PECAM and CD99, L2 promotes transmigration by recruiting the lateral border recycling compartment to sites of TEM, specifically downstream of PECAM initiation. Collectively, our data identify a novel functional role for human L2 in TEM and elucidate a mechanism that is distinct from PECAM and CD99.

Role of CD99 in regulating homeostasis and differentiation in normal human epidermal keratinocytes.

CD99 is a glycoprotein primarily expressed in immune cells. Physiologically, it is involved in the adhesion, migration, and development of immune cells. The presence of CD99 in the skin was first reported in 2016 and its function is yet to be determined. In this study, we aimed to understand the role of CD99 in the skin using normal human epidermal keratinocytes (NHEK). CD99 expression increased with the confluency of NHEK, while the CD99-high expressing NHEK lost their stem cell properties and played a role in barrier function. We characterized CD99-expressing NHEK as cells committed to early differentiation because they expressed early differentiation markers. However, the deficiency of CD99 in NHEK disrupted homeostasis and caused aberrant differentiation, as evidenced by larger cells with lesser Ki67 staining and higher expression of terminal differentiation markers. Hence, we propose that CD99 is involved in maintaining homeostasis and initiating early differentiation in the skin.

Does PAX7 and NKX2.2 immunoreactivity in Ewing sarcoma have prognostic significance?

Ewing sarcoma (ES) is an aggressive neoplasm with variable morphology. It has no specific immunoprofile or molecular signature. Neither CD99, NKX2.2 nor PAX7 immunoreactivity alone is completely specific, although diagnostic specificity improves when combined. The purpose of the present study was to investigate the immunohistochemical (IHC) expression of PAX7 in a large series of genetically confirmed ES. Existing results for CD99 and NKX2.2 immunoexpression, morphological findings and molecular studies (fusion gene subtypes) were retrieved from a previous study. Survival analyses were performed in cases with available clinical follow-up. PAX7 was positive in 95.5% of ES with diffuse staining (> 50%) in all positive cases and moderate or strong intensity for most cases. Nineteen ES displayed both PAX7 and CD99 immunoreactivity but lacked NKX2.2 immunoexpression. No relationships could be found between PAX7 expression and the histological types or ES gene fusion subtypes. Univariant/multivariate analysis showed that lack of PAX7 and/or NKX2.2 immunoexpression constitute independent poor prognostic factors for progression free survival (PFS) and overall survival (OS). In conclusion, IHC for CD99, NKX2.2, and PAX7 may be useful in daily practice for ES diagnosis, particularly in hospitals lacking facilities for molecular studies. In addition, the combination of strong CD99 membranous positivity and nuclear PAX7 and NKX2.2 immunoreactivity seems to be very reliable for ES diagnosis when supported by a corroborating histomorphologic and clinical picture. Although PAX7 is not entirely specific for ES, it seems to have a more extensive and strong nuclear immunoreactivity than NKX2.2 expression, even in tumors with decalcification artifact. Considering the prognostically significant data herein reported, we strongly recommend validation in prospective ES series that include localized and disseminated tumors.

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.

Targeting CD99 Compromises the Oncogenic Effects of the Chimera EWS-FLI1 by Inducing Reexpression of Zyxin and Inhibition of GLI1 Activity.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.