Abstract 2868: CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways

Ewing sarcoma (ES) cells express high levels of the cell surface protein CD99, which is used for diagnosis of ES. More importantly, the inhibition of CD99 activity results in reduced growth of ES cells in vitro and in vivo. In an earlier publication, we have identified clofarabine and cladribine as selective inhibitors of CD99, which inhibited ES growth both in vitro and in vivo. Clofarabine and cladribine directly bound to CD99, inhibited its molecular interactions, and regulated CD99 specific intracellular signaling pathways. In order to gain further insight into how clofarabine may regulate cellular functions through inhibiting CD99, we utilized a phospho-kinase array to identify changes in phosphorylation levels of 43 proteins in response to clofarabine or CD99 antibody treatment. We identified ERK1/2-MSK1/2-CREB signaling axis as one of the signaling pathways downstream of CD99. These findings were validated in four different ES cell lines and in xenograft lysates that were harvested from clofarabine-treated mice compared to control mice. The phosphorylation events induced by clofarabine treatment was significantly diminished when CD99 was knocked down either by siRNA-mediated knockdown or CRISPR/Cas9-mediated genomic disruption. Time-course experiments on ES cells showed that clofarabine triggers a very rapid signaling cascade through CD99. Cytarabine, a structurally similar pyrimidine analog and an inhibitor of the EWS-FLI1 transcriptional activity that has been failed in clinical trials on ES patients, did not activate ERK1/2, MSK1/2 or CREB phosphorylation in ES cells. In order to test whether the observed changes in phosphorylation levels are required for cell death, we screened a kinase inhibitor small molecule library for their ability to rescue clofarabine-induced death of ES cells. We identified TG100-115 (PI3K inhibitor) and rebastinib (c-abl inhibitor) as the most potent kinase inhibitors that showed significant reversal of clofarabine-induced ES cell death, suggesting that clofarabine-induced death of ES cells requires specific phosphorylation cascades. We then discovered that CD99 can be co-immunoprecipitated with FGFR1 and clofarabine treatment of ES cells results in phosphorylation of FGFR1. In conclusion, we discovered a novel mechanism of action for clofarabine and cladribine in that their selective cytotoxic effect on ES is through inhibiting CD99 on the cell surface, and it is not dependent on their ability to inhibit DNA synthesis. CD99 inhibition resulted in increased phosphorylation of FGFR and two downstream signaling pathways (PI3K/Akt and MEK1/ERK1). Inhibition of PI3K pathway rescues clofarabine-induced ES cell cytotoxicity. These findings provide additional mechanistic support for repurposing clofarabine and cladribine for ES indication. Note: This abstract was not presented at the meeting. Citation Format: Haydar Celik, Levent Dusunceli, Anna Molotkova, David V. Allegakoen, Erin J. Conn, Jeff R. Petro, Jeffrey A. Torestky, Aykut Uren. CD99 inhibition by clofarabine induces Ewing sarcoma cytotoxicity through activation of FGFR1 and downstream kinase pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2868.