Ewing sarcoma (ES) is the second most common bone tumor in the pediatric population and is defined by a pathognomonic translocation, most frequently EWS::FLI1, which leads to the expression of a novel, yet not presently targetable oncogenic transcription factor. The treatment regimen for ES includes cytotoxic chemotherapy, surgery and/or radiation, which have improved overall survival rates to approximately 70%. However, despite intensification of therapeutic regimens, long-term outcomes for patients with metastatic or relapsed Ewing sarcoma remain extremely poor, with overall survival rates dropping to 20-30%. Thus, alternative targeted therapies, or combination therapies, are essential for improving patient outcomes. This proposal addresses a significant gap in the knowledge towards treatment for high-risk ES. Our laboratory has recently identified p21-activated kinases (PAKs) are activated and promote metastasis in ES. PAKs are a family of serine/threonine kinases that regulate key cell processes involved in metastatic progression and drug resistance and are imminently emerging as candidate anti-cancer targets. We recently reported that PAK4, which has oncogenic properties and is a nidus for critical signaling cascades including Ras/MAPK, Wnt, and PI3K signaling, is significantly overexpressed in metastatic ES, and is negatively correlated with patient outcomes. In vitro and in vivo molecular and pharmacological perturbations of PAK4 in ES models demonstrated significant anti-tumor and anti-metastatic activity. In addition, we have preliminary evidence that PAK4 is a novel regulator of alternative splicing and subsequently modulates EWS::FLI1 activity. Using our innovative, state-of-the-art approaches and unique in vivo and ex vivo models, the overarching goals are to gain additional molecular insights into PAK4- mediated mechanisms in ES and investigate the therapeutic potential of targeting PAK4. We will examine PAK4’s role as a novel regulator of alternative splicing and PAK4-mediated downstream proteo-transcriptomic signatures that contribute to ES progression. We will also investigate and assess the efficacy of biologically rational and clinically applicable synergistic therapeutic regimens. We hypothesize that PAK4 integrates signaling events critical for ES development and progression and regulates novel downstream effectors, including spliceosome components that significantly contributes to its oncogenic properties. Our aims include the following: Aim1. Defining the PAK4 direct substrates and functional subcellular signaling networks in primary and metastatic Ewing sarcoma. Aim2. Evaluation of PAK4 as a novel splicing regulating protein kinase and its direct effects on EWS::FLI1 function and downstream gene regulation in ES. Aim3. Assess the therapeutic potential of inhibiting PAK4 in combination with mechanistically rational targeted agents using orthotopic and metastatic ES models. Completion of these studies will provide new insight...