Objective EWS‐FLI1 is the most common oncogenic fusion protein in Ewing's sarcoma family tumors (ESFTs). DAX1, an orphan member of the nuclear receptor superfamily, is up‐regulated by EWS‐FLI1 and plays a key role in the transformed phenotype of ESFTs. Methods To discover a functional inhibitor of DAX1 and EWS‐FLI1, we screened small‐molecular inhibitors using a DAX1 reporter assay system. Results K‐234 and its derivatives, which were dihydroorotate dehydrogenase (DHODH) inhibitors, showed inhibitory effects in the reporter assay. K‐234 inhibited the growth of Ewing's sarcoma with various fusion types, and K‐234 derivatives altered the expression of EWS‐FLI1‐regulated genes. The DAX1 expression had no effect on the growth inhibitory effect of the K‐234 derivatives, while DHODH overexpression or uridine treatment attenuated their inhibitory effects, suggesting that inhibition by K‐234 derivatives occurs through DHODH inhibition. An in vivo study showed that a K‐234 derivative clearly inhibited tumor growth in an Ewing's sarcoma xenograft mouse model. Conclusion Taken together, the present results suggest that DHODH inhibitors can inhibit the function of DAX1/EWS‐FLI1 in ESFTs and might be a therapeutic agent with potent anti‐tumor activity for Ewing's sarcoma patients.

Background: Ewing's sarcoma family of tumors (ESFTs) are characterized by chromosomal translocations that fuses EWSR1 gene and other types of ETS family genes. Among them, EWS-FLI1 is the most common transcriptional factor which regulates many genes of biological pathways leading to cell cycle, metabolic and DNA repair. EWS-FLI1 oncoprotein is an ideal therapeutic target for ESFTs whereas it turned out to be difficult to obtain direct small-molecule inhibitor of EWS-FLI1 due to lack of intrinsic enzymatic activity. Therefore, we postulate indirect inhibitors of EWS-FLI1 function that can suppress the transcriptional activity of EWS-FLI1, resulting in selectively inhibition of growth of ESFTs. Results: To discover small molecular compounds which inhibit the cell growth in Ewing's sarcoma cells, a cell proliferation assay using Ewing's sarcoma A-673 cells harboring EWS-FLI1 fusion protein was performed. We identified compound A which inhibited the cell proliferation in A-673 and Ewing's sarcoma TC-71 cells with GI50 values of 27 nM and 25 nM, respectively. Compound A also inhibited colony formation of all some Ewing's sarcoma cells. In contrast, growth inhibition by compound A in pancreatic AsPC-1 cells which express no EWS-FLI1 was not observed at the concentration up to 10000 nM. A derivative of compound A as well as EWS-FLI1 siRNA decreased the expression of NKX2.2 and CCND1, and increased the expressions of IGFBP3, PHLDA1 and DKK1. These genes are under the downstream control of EWS-FLI1 so that compound A might down-modulate EWS-FLI1 function. We found that a series of derivatives inhibited the enzymatic activity of nucleotide biosynthesis. IC50 values of the enzyme inhibitory activities among derivatives were correlated well with GI50 values of anti-proliferative activities in A-673 cells (r = 0.86). Moreover, overexpression of the enzyme gene in A-673 cells could attenuate the anti-proliferative activities of the derivatives, suggesting that inhibition of the enzyme by compound A is involved in the down-modulation of EWS-FLI1 driven growth. Finally, using a Ewing's sarcoma xenograft mouse model, oral daily administration of the derivative at 100 mg/kg considerably inhibited the tumor growth with a minimum T/C ratio of 0.13 without body weight loss. Conclusions: Compound A and its derivatives may be a therapeutic agent with potent antitumor activity for Ewing's sarcoma patients. Citation Format: Hiromichi Kosaka, Yasuo Watanabe, Michihiro Maemoto, Masamori Sugawara, Miwa Watanabe, Yoko Ono, Yoshisuke Nakasato, Masahiro Matsubara, Ryuichiro Nakai. Small molecule metabolic inhibitors, compound A and the derivatives specifically inhibit the cell growth of Ewing's sarcoma cells harbor EWS-FLI1 in vitro and in vivo. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A155.