Ewing sarcoma is a bone and soft tissue sarcoma that occurs in children and young adults. The EWS-FLI1 gene fusion is the driver mutation in most Ewing sarcoma tumors and functions, in part, as an aberrant transcription factor. We recently identified that Ewing sarcoma cells are sensitive to inhibition of ribonucleotide reductase (RNR), which catalyzes the formation of deoxyribonucleotides from ribonucleotides. In this report, we show that Ewing sarcoma cells are sensitive to treatment with clofarabine, which is a nucleoside analogue and allosteric inhibitor of RNR. However, clofarabine is a reversible inhibitor of RNR and we found that the effect of clofarabine is limited when using a short (6-hour) drug treatment. Gemcitabine, on the other hand, is an irreversible inhibitor of the RRM1 subunit of RNR and this drug induces apoptosis in Ewing sarcoma cells when used in both 6-hour and longer drug treatments. Treatment of Ewing sarcoma cells with gemcitabine also results in activation of checkpoint kinase 1 (CHK1), which is a critical mediator of cell survival in the setting of impaired DNA replication. Notably, inhibition of CHK1 function in Ewing sarcoma cells using a small-molecule CHK1 inhibitor, or siRNA knockdown, in combination with gemcitabine results in increased toxicity both in vitro and in vivo in a mouse xenograft experiment. Overall, our results provide insight into Ewing sarcoma biology and identify a candidate therapeutic target, and drug combination, in Ewing sarcoma.

There are no universally approved re-vaccination guidelines for non-transplant pediatric cancer survivors. We hypothesized that by utilizing a response-based re-vaccination schedule, we could tailor vaccine schedules in off-treatment cancer survivors. Pre-vaccination antibody levels were obtained in 7 patients at an average of 20 days after the end of treatment date. In those without protective antibody levels, we administered vaccines 3 months after completion of treatment. Revaccinating patients 3 months after the end of treatment date resulted in protective antibody levels for most vaccines. We showed, on a preliminary basis, that vaccinating non-transplanted pediatric cancer survivors can be dynamically implemented in children with recovering immune function.

Ewing sarcoma is a highly aggressive bone and soft tissue cancer that is caused by the EWS-FLI1 fusion protein. The EWS-FLI1 oncoprotein functions, in part, as an aberrant transcription factor and is required for tumor growth and survival. In order to identify downstream targets of EWS-FLI1, we used human embryonic stem cells that express inducible EWS-FLI1 to model the initiation and development of Ewing sarcoma in a genetically defined system. We then used this model system and a gene expression based approach to identify that Ewing sarcoma cells are uniquely vulnerable to inhibitors of ribonucleotide reductase (RNR), which impair DNA replication by blocking the synthesis of deoxyribonucleotides. Here we report that the treatment of Ewing sarcoma cells with gemcitabine, an irreversible inhibitor of the RRM1 subunit of RNR, results in impaired DNA replication, cell cycle arrest, and apoptosis in Ewing sarcoma cells. Additionally, we have found that the effect of gemcitabine on the viability of Ewing sarcoma cells is sustained even after removal of the drug from the cell culture medium. Moreover, ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (CHK1) inhibitors increase the toxicity of gemcitabine in Ewing sarcoma cells by blocking the adaptive response to impaired DNA replication. Currently, ongoing work is focused on the in vivo testing of gemcitabine, alone and in combination with CHK1 and ATR inhibitors, as a novel therapeutic approach for the treatment of Ewing sarcoma. Citation Format: Kelli Goss, Stacia Koppenhafer, Kathryn Harmoney, David Gordon. Targeting ribonucleotide reductase (RNR) in 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 1955. doi:10.1158/1538-7445.AM2017-1955