Abstract 2743: Targeting Ewing sarcoma with a new small molecule binding to the EWS-FLI1 oncoprotein.
Authors: Kaluz Stefan , Yin Shaoman , Devi Sarojini N. , Van Meir Erwin G. 
Published: August 2014

Ewing Sarcoma (ES) is a rare malignant round-cell tumor growing in bone or soft tissue that occurs in childhood or early adulthood. Patients with ES have a high rate of tumor recurrence and poor prognosis, highlighting the urgent need for new therapies. About 95% of ES harbor a t(11;22)(q24;q12) chromosomal translocation, resulting in an in-frame gene fusion between the EWSR1 gene (chr.22) and one of the ETS family genes, mainly FLI1 (chr.11). Recent studies show that EWS-FLI1 is an oncoprotein that plays a central role in ES tumor initiation and development. Given the specificity and critical role of EWS-FLI1 in ES tumorigenesis, identification of new compounds that bind EWS-FLI1 is of great significance for the development of new therapeutic approaches for ES. Unfortunately, the rational design and screening of EWS-FLI1- targeting small molecules has been hampered due to the poor solubility of the protein in vitro and limited structural information.

Here we report the results of a small molecule screening aimed at identifying molecules cytotoxic to ES cells, but not to glioma or non-tumoral control cells. In this screening, we identified a class of sulfonamides that consistently yielded ES cell inhibitors. KCN1, the lead compound, significantly decreased viability of ES cells but not control cells in vitro, as demonstrated by sulforhodamine B and clonogenicity assays. KCN1 blocked progression through the cell cycle and triggered apoptosis in ES cells. Expression profiling revealed that KCN1 prominently upregulated expression of genes coding for enzymes of the mevalonate (cholesterol synthesis) pathway, suggesting metabolic reprogramming. Further, intraperitoneal administration of KCN1 (60 mg/kg) in ethanol/cremophor formulation 5 times/week inhibited the growth of ES cells in vivo in a nu/nu mouse model, while being very well tolerated without any overt toxicity. To identify the molecular target of KCN1, we used an affinity purification approach with a KCN1-biotin probe on cell extracts. Following mass spectrometry, we identified that KCN1 interacts directly with EWS and EWS-FLI1, suggesting binding to EWS N-terminus. ES cell treatment with KCN1 is accompanied by downregulation of EWS-FLI1 transcriptional targets (NR0B1, cyclin D1, GLI1) and further characterization of its mechanisms is ongoing.

In summary, we identified arylsulfonamides as a novel class of agents with anti-ES activity. We are the first to report identification of a small chemical molecule inhibitor directly interacting with the N-terminus of the EWS-FLI1 fusion protein (first-in-class). KCN1 inhibits the growth of ES cells in vitro and in vivo. Considering the lack of targeted therapies and the apparent non-toxic nature of KCN1, our data warrant further preclinical and clinical testing of KCN1 and other molecules in the arylsulfonamide class for treating ES as well as further study of their precise mechanism of action.

Citation Format: Stefan Kaluz, Shaoman Yin, Sarojini N. Devi, Erwin G. Van Meir. Targeting Ewing sarcoma with a new small molecule binding to the EWS-FLI1 oncoprotein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2743. doi:10.1158/1538-7445.AM2013-2743

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.