Abstract 3412: Phage display identifies novel toxic binding peptides for the disordered oncoprotein EWS-FLI1
Authors: Lauren M. Jacobwitz, Hayriye V. Erkizan, Aykut Üren, Jeffrey A. Toretsky
Journal: Cancer Research (2010)

Abstract Introduction: The EWS-FLI1 oncogenic fusion protein is derived from a tumor-specific t(11;22) uniquely expressed in Ewing's Sarcoma Family Tumors (ESFT). EWS-FLI1 is a disordered protein and thus presents opportunities for manipulating protein-protein interactions, some of which will be specific anti-tumor targets. We previously discovered and targeted RNA Helicase A as a key protein partner of EWS-FLI1 using data from a phage display experiment. Phage display is a powerful technique to identify peptides that bind to disordered proteins, such as EWS-FLI1. We hypothesize that by identifying additional peptides and their cognate proteins, we can investigate the function of EWS-FLI1 to improve therapies for ESFT patients. Methods: Direct binding to EWS-FLI1 was analyzed using ELISA. MTT assays were used to determine cytotoxicity. Peptides identified from phage were searched using NCBI BLAST and estimates of disordered regions were made using PONDR. Results: Phage display 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. These 27 peptides were commercially synthesized with the Antennapedia sequence and evaluated in TC32 (EWS-FLI1, type 1), JN-DSRCT-1 (desmoplastic small round cell tumor containing EWS-WT1), and SKNAS (neuroblastoma cell line lacking EWS-FLI1). At 30 microM concentration 6 out of 27 peptides inhibited growth &amp;gt;50% in TC32, 17 out of 27 peptides inhibited growth &amp;gt;50% in JN-DSRCT-1, and 0 out of 27 peptides inhibited growth &amp;gt;50% in SKNAS. We identified the A1 peptide (sequence: TMRGKKKRTRAN) and cell lines were treated with 30 microM with survival at 7% in TC32, &amp;lt;5% in JN-DSRCT-1, and &amp;gt;75% in SKNAS after four days. 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. A BLAST search with the A1 sequence against known proteins in the NCBI database resulted in four proteins with homologies ranging between 33%-58%: MLL3, DDX27, BRD9, and TXNDC9. Preliminary data from co-immunoprecipitation confirms the presence of BRD9 in TC32 cells and suggests that it participates in a previously undescribed protein complex with EWS-FLI1. Discussion: The A1 peptide appears to directly bind to the EWS-FLI1 oncoprotein and leads to cell death in cells with either the EWS-FLI1 or EWS-WT1 fusion proteins. Further work will include additional analyses of A1 binding to the oncogenic fusion proteins, minimal peptide sequences for cell toxicity and inhibition of oncogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3412.