Understanding the Effects of Phosphorylation on the Structural Dynamics of EWS‐FLI1 and its Self‐Associative Behavior

Abstract

Ewing sarcoma is a malignant tumor that occurs in bones or soft tissues, such as cartilage or nerves. Ewing sarcoma is a rare cancer occurring with a global frequency of approximately 1.5 cases per million children and adolescents. Ewing tumors are treatable if diagnosed early, but their aggressiveness leads to metastasis or relapse, upon which the survival rate is reduced to less than 30 percent. Development of targeted therapies are urgently needed since the current standard of care using repurposed adult cancer therapeutics are extremely toxic leaving juvenile patients with lifelong debilities and reduced lifespan. Ewing sarcoma is characterized by a chromosomal translocation, most commonly t(11;22)(q24;q12), which joins the low-complexity domain of the RNA-binding protein Ewing sarcoma breakpoint region 1 (EWSR1) with DNA binding domain of the transcription factor, Friend Leukemia Integration 1 (FLI1). The resultant oncogenic fusion protein, Ewing sarcoma-Friend Leukemia Integration 1 (EWS-FLI1) is considered an intrinsically disordered protein (IDP), a class of proteins that are functional, yet lack stable secondary or tertiary structures. The structural dynamics and functions of IDPs are frequently associated with post-translational modification (PTM) sites, which serve to regulate biological functions such as biomolecular interactions or sub-cellular localization. By analogy with Fused in Sarcoma (FUS), a closely related family member of EWSR1, we have identified 8 DNA PK (S/TQ) target sites in the EWS portion of EWS-FLI1. Because protein phosphorylation is used as a mechanism to control many life critical processes, we are hypothesizing that phosphorylation of EWS-FLI1 is an important modulator for its activity. Using an in vitro phosphorylation assay, we have shown that DNA PK phosphorylates several of these sites. We are now interested in observing the structural and biophysical impacts of phosphorylation on EWS-FLI1 including the potential modulation of its self-associative function. We will use NMR and mass spectroscopy to identify the phosphorylation sites, determine the frequency of modification and assess the impact of this PTM on the structure of EWS-FLI1. Finally, using S/T to E as phosphomimetic mutations we will examine the impact of phosphorylation on the self-associative function of EWS-FLI1 using fluorescence-based aggregation assay. If successful, this will help us to understand the function of EWS-FLI1's self-associative behavior and identify novel therapeutic vulnerabilities.