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Treatment failure in high risk neuroblastoma (NB) is largely due to the development of chemotherapy resistance. We analyzed the gene expression changes associated with exposure to chemotherapy in six high risk NB tumors with the aid of the Connectivity Map bioinformatics platform. Ten therapeutic agents were predicted to have a high probability of reversing the transcriptome changes associated with neoadjuvant chemotherapy treatment. Among these agents, initial screening showed the EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279, had obvious cytotoxic effects on NB cell lines. Using a panel of NB cell lines, including MYCN nonamplified (SK-N-AS, SH-SY5Y, and CHLA-255), and MYCN amplified (NB-19, NGP, and IMR-32) cell lines, we found that YK-4-279 had cytotoxic effects on all lines tested. In addition, YK-4-279 also inhibited cell proliferation and anchorage-independent growth and induced cell apoptosis of these cells. YK-4-279 enhanced the cytotoxic effect of doxorubicin (Dox). Moreover, YK-4-279 was able to overcome the established chemoresistance of LA-N-6 NB cells. In an orthotopic xenograft NB mouse model, YK-4-279 inhibited NB tumor growth and induced apoptosis in tumor cells through PARP and Caspase 3 cleavage in vivo . While EWS-FLI1 fusion protein is not frequently found in NB, using the R2 public database of neuroblastoma outcome and gene expression, we found that high expression of EWSR1 was associated with poor patient outcome. Knockdown of EWSR1 inhibited the oncogenic potential of neuroblastoma cell lines. Taken together, our results indicate that YK-4-279 might be a promising agent for treatment of NB that merits further exploration., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Ewing sarcoma is an aggressive tumor of bone and soft tissue affecting predominantly children and young adults. Tumor-specific chromosomal translocations create EWS-FLI1 and similar aberrant ETS fusion proteins that drive sarcoma development in patients. ETS family fusion proteins and over-expressed ETS proteins are also found in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Transgenic expression of EWS-FLI1 in mice promotes high penetrance erythroid leukemia with dense hepatic and splenic infiltrations. We identified a small molecule, YK-4-279, that directly binds to EWS-FLI1 and inhibits its oncogenic activity in Ewing sarcoma cell lines and xenograft mouse models. Herein, we tested in vivo therapeutic efficacy and potential side effects of YK-4-279 in the transgenic mouse model with EWS-FLI1 induced leukemia. A two-week course of treatment with YK-4-279 significantly reduced white blood cell count, nucleated erythroblasts in the peripheral blood, splenomegaly, and hepatomegaly of erythroleukemic mice. YK-4-279 inhibited EWS-FLI1 target gene expression in neoplastic cells. Treated animals showed significantly better overall survival compared to control mice that rapidly succumbed to leukemia. YK-4-279 treated mice did not show overt toxicity in liver, spleen, or bone marrow. In conclusion, this in vivo study highlights the efficacy of YK-4-279 to treat EWS-FLI1 expressing neoplasms and support its therapeutic potential for patients with Ewing sarcoma and other ETS-driven malignancies.