The overarching goal of this proposal is to understand protein complexes and conformations that maybe targetable in Ewing sarcoma (ES), the second most-common pediatric bone cancer. ES is aggressively metastatic with no targeted therapies. A lack of protein structural information for the EWSFLI1fusion of a transcription factor to an intrinsically disordered domain stymies design of targeted therapies. However, metastatic cells definably expressing low levels of the fusion oncoprotein EWSFLI1.

Promising therapies cannot be assessed in distinct proliferative (high EWS-FLI1) and metastatic (low EWS-FLI1), or drug-resistant cells as targets include nu...

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The overarching goal of this proposal is to understand protein complexes and conformations that maybe targetable in Ewing sarcoma (ES), the second most-common pediatric bone cancer. ES is aggressively metastatic with no targeted therapies. A lack of protein structural information for the EWSFLI1fusion of a transcription factor to an intrinsically disordered domain stymies design of targeted therapies. However, metastatic cells definably expressing low levels of the fusion oncoprotein EWSFLI1.

Promising therapies cannot be assessed in distinct proliferative (high EWS-FLI1) and metastatic(low EWS-FLI1), or drug-resistant cells as targets include numerous protein complexes lacking selective probes involved in metastasis (e.g., cytoskeletal complexes targeted by PAK1 inhibition)and/or proliferation (e.g., HSP90 chaperone-client protein complexes).

This proposal bolsters multi-pronged rational evaluation of targets and therapeutics for ES by innovating micro-engineered systems for isolation of:

1. Protein complexes from single cells
2. Molecular conformation of disordered protein

To evaluate the response of cytoskeletal protein complexes (e.g., focal adhesions) to PAK1-inhibition in metastatic ES subpopulations, we introduce microparticle tumor models mimicking the bone microenvironment to targeted single-cell protein complex electrophoretic separations. In a separate aim we will discover chaperone-client protein complexes that exist in ES cell subpopulations designing protein-complex separations and protein collection for single-cell mass spectrometry. Finally, we will identify structural elements of the disordered EWS-FLI1 by constraining molecular conformational heterogeneity in a nano-gel-embedded cryoEM grid.

The suite of protein technologies will have wide-ranging applications in the study of metastasis, chaperone complexes and fusion oncoprotein structures across numerous cancers.

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