PROJECT SUMMARY/ABSTRACT Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. Overall, survival rates for RMS are approximately 65-70%. However, despite intensification of chemotherapeutic regimens, long-term outcomes for patients with metastatic or relapsed rhabdomyosarcoma remain extremely poor, with overall survival rates between 20-30%. Thus, alternative targeted therapies are essential for improving patient outcomes. Our proposal will address a significant gap in the knowledge and treatment of high-risk rhabdomyosarcoma. RMS tumors have frequent mutations in the RAS/NF1 pathway and perturbations in the receptor tyrosine kinases (RTK)/RAS/phosphoinositide 3-kinase (PI3K) signaling axis, which are associated with high-risk conditions. The p21-activated kinases (PAK) family of serine/threonine kinases intersects the oncogenic signaling by RTK and RAS-activated pathways. Downstream PAK signaling regulates metastatic phenotypes and resistance to therapeutics, thus making them attractive targets for advanced cancers. Based upon extensive informatic, functional genomic, biochemical interactome and pharmacological data we have evidence that PAK4 regulates intrinsic and extrinsic mechanisms, including alternative splicing(AS) and immune modulatory signals, that contributes to RMS progression. In addition, using high-throughput combination drug screens we identified mechanistically rational and clinically applicable synergistic therapeutic regimens with PAK4 inhibition. The overarching goals of our proposal are to gain critical molecular insights into novel PAK4-mediated mechanisms and further investigate the therapeutic potential of targeting PAK4 in RMS. We will examine PAK4’s role as a novel regulator of AS and PAK4-mediated downstream transcriptomic and proteomic signatures that contribute to RMS progression through the use of our extensive inventory of highly relevant human and syngeneic murine pre-clinical models. We hypothesize PAK4 regulates critical intrinsic and extrinsic molecular signatures vital for RMS progression, and PAK4 inhibitors in combination with rational targeted agents can provide effective novel therapeutic regimens for these high-risk patients. We propose three independent, yet innovative and novel complementary aims to address this hypothesis. Aim1. Evaluation of PAK4 as a novel splicing regulating protein kinase and its direct effects on downstream signaling in RMS. Aim 2. Role of PAK4 in modulating the tumor microenvironment and identification of therapeutic applications using relevant murine pre-clinical models of RMS developed in our lab. Aim3. Investigate the therapeutic potential of inhibiting PAK4 in combination with mechanistically rational targeted agents using innovative ex vivo 3D lung metastasis models and in vivo orthotopic and metastatic RMS models. Completion of these studies will provide novel molecular insights into RMS biology and impact RMS therapy by providing new the...