Abstract 259: Novel peptide directed targeting of EWS-FLI1 impairs tumor cell growth

Abstract

Abstract Introduction: Patients with Ewing's Sarcoma require improved therapy. EWS-FLI1 fusion protein provides a unique targeting opportunity. In the quest for protein partners, we identified a novel peptide sequence that binds to EWS-FLI1 using phage display. We hypothesize that by identifying peptides, and their cognate proteins, we can modulate the function of EWS-FLI1 to improve therapies for ESFT patients. Methods: Secreted peptide-alkaline phosphatase conjugates were used in ELISA for resolving binding regions. Cytotoxicity experiments were analyzed with MTT or WST reduction. Cell cycle analysis were performed using flow cytometry. Results: A phage display experiment to identify EWS-FLI1 peptides resulted in 96 phage clones that demonstrated binding to EWS-FLI1 by ELISA. Those 96 phage clones resulted in sequences that yielded twenty-seven unique peptides. Peptide A1 is one of 27 unique peptides that was commercially synthesized fused to penetratin (16aa) to facilitate intracellular transport. The cytotoxicity of A1 peptide was evaluated in TC32 and TC71 (EWS-FLI1, type I), A4573 (EWS-FLI1, type III), JN-DSRCT (desmoplastic small round blue cell tumor containing EWS-WT1), and SKNAS (a neuroblastoma cell line lacking EWS-FLI1 rearrangement). The IC50 level for A1 is between 5 -15 μM in cell lines bearing EWS fusion proteins. However, it is more than 50 μM in cells without the rearrangement. A1-alkaline phosphatase (A1-AP) conjugates were prepared with alanine substitutions throughout the peptide. Wild-type non-conjugated A1 competed away the interaction between the EWS-FLI1 and A1-AP, confirming its ability to bind to EWS-FLI1. Mutations in A1 demonstrated that the lysine residues are critical for binding to EWS-FLI1. The minimal interacting amino acids were validated using shortened and mutated versions of A1 in both ELISA and cell growth assays, including soft agar. A1 peptide blocked cell cycle progression of synchronized ESFT cells and caused accumulation at S phase. A1 peptide also inhibited EWS-FLI1 dependent transcription. Discussion: The A1 peptide directly binds to the EWS-FLI1 oncoprotein and leads to cell death in cells with either the EWS-FLI1 or EWS-WT1 fusion proteins. While A1 was discovered based on binding to EWS-FLI1; toxicity in JN-DSRCT studies suggest that the A1 peptide may have potency to EWS bearing fusion proteins. These findings present a novel method of targeting EWS rearranged oncoproteins and could lead to novel therapies Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 259. doi:10.1158/1538-7445.AM2011-259