Designing inhibitors to a key protein interaction domain of oncogenic EWS-FLI1

Research project P 13708 EWS-FLI1 inhibitors Heinrich KOVAR 28.06.1999 ETYS-ets gene rearrangements are a hallmark of the Ewing family of tumors (EFT). They encode chimeric transcription factors whose continued expression is required for tumor cell growth and tumorigenicity. In a preceding project supported by the FWF (P-12261GEN) we identified the general transcription factor hsRPB7 as a nuclear protein specifically interacting with the N-terminal EWS domain of the rear-ranged ets-transcription factor but not of germline EWS. It thus appears that replacement of the EWS C-terminus by a DNA-binding transcription factor moiety results in a conformational switch at the N-terminus exposing an otherwise unaccessible protein interaction domain for communication with hsRPB7 and possibly other cellular Proteins. This project proposal aims at the generation of agents that disrupt protein interaction at the EWS N-terminus to compromise EWS-ets function in EFT. After fine mapping of the protein interaction interface peptides shall be synthesized that will be tested for their ability to compete with EWS-ets / hsRPB7 interaction. Such peptides will be coupled to Penetratin to allow for efficient non-traumatic cellular internalization. EFT and non-EFT cells will be analysed for their growth/apoptosis characteristics as well as immunophenotype in the presence and absence of the peptides. In addition, biologically functional peptides will be used to generate a monospecific monoclonal antibody (mAb). This agent will be tested for reactivity with rearranged as compared to germline EWS. Hopefully, it may turn out as an EFT specific immunological reagent for routine pathohistological evaluation. The light and heavy chain variable regions for this mAb will be cloned to generate a recombinant single chain antibody (sFv) as a basis for the construction of a mammalian vector encoding an intracellularly expressed sFv (intrabody). Suitable modifications will allow nuclear expression and direct microscopic monitoring of intrabody expression in living cells. The potential of this reagent to specifically inhibit EFT cell growth will be extensively evaluated. These studies may constitute a first step towards innovative biological therapy of EFT.

We have generated in-vitro single chain antibodies targeting a tumor specific nuclear oncoprotein that can be expressed within the tumor cell in order to interfere with its cellular oncogenic function. Tumor-associated chromosomal rearrangements found in Ewings sarcoma family tumors (EFT) fuse part of the EWS gene to one of 5 members of the ets family of transcription factor genes (FLI1, ERG, ETV, FEV, and E1AF). The EWS-ETS fusion products have been shown to play a dominant role in EFT pathogenesis. In this project we aimed to design specific inhibitors to the oncogenic transcription factor EWS-FLI1, arising from a translocation between chromosomes 11 and 22 and expressed in about 85% of EFT. These agents should target a specific epitope on EWS-FLI1 and interfere with protein-protein interactions mediated by this domain. The project was based on the observation that the RNA polymerase subunit hsRPB7 specifically interacts with EWS-FLI1 but not with germline EWS although the interacting domain is present on both proteins, implicating that an epitope not exposed on germline EWS gets accessible to protein interactions as a consequence of the gene fusion to FLI1. We have delineated the minimal communication epitope for hsRPB7 and planned to design competing peptides mimicking the interacting epitopes and intracellular single-chain antibodies (intrabodies) to the interaction surface as two alternative approaches to inhibit EWS-FLI1 activity. The cell-permeable peptide approach was impeded by our inability to establish a robust in-vitro physical interaction assay for hsRPB7 with EWS-FLI1 due to the weakness of this interaction. Using synthetic recombinant antibody phage display libraries, we generated several anti-EWS- FLI1 single-chain antibodies. The clones that gave the highest value in an immunological test bind recombinant EWS-FLI1 protein with high affinity but, so far, showed no reactivity towards endogenous cellular EWS-FLI1 in in-vitro assays. Still ongoing studies should establish whether these molecules interact with EWS-FLI1 in vivo. In addition, our protein interaction studies in the course of this project not only identified a novel interaction between EWS-FLI1 and the tumor suppressor BARD1 but also the potential to oligomerize. We have obtained preliminary evidence for a role of post-translational protein modification in the potential of EWS-FLI1 to interact with itself and possibly with other proteins including the single chain antibodies created in this study. Future investigations will therefore concentrate on the characterization and regulation of these potentially biological important modifications.