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Proposal: Rhabdomyosarcoma (RMS), the predominant soft-tissue childhood sarcoma, is a devastating solid tumor with myogenic features. Despite a generally aggressive treatment, less than 25% of patients with metastatic RMS achieve 5- year survival. The rewiring of metabolic programs to meet the demands of growth and proliferation is a characteristic hallmark of cancer, largely relies on a hierarchical oncogenic cascade involved in Akt/mTOR, MAPK and essentially a HIF1α- or Myc-dependent metabolic transcriptome. However, the metabolic rewiring and its contribution to the pathogenesis of RMS are incompletely understood. The cross-referencing of the microarray data of RMS from multiple resources revealed an enriched metabolo-transcriptome signature linked to the metabolism of γ-aminobutyric acid (GABA), a known neurotransmitter. GABA is the major inhibitory neurotransmitter in the central nervous system (CNS) and the dysregulation of GABA metabolism and signaling is associated with a number of neurological disorders, including epilepsy and schizophrenia. However, recent accumulating evidence suggested that GABA is also synthesized in non-neuronal cells and may act as a trophic factor contributing the various physio-pathological processes. We have validated that the ratelimiting enzyme in GABA metabolism, GABA transaminase (ABAT), is highly upregulated in a set of primary RMS tumor samples (from both human and mouse) and RMS cell lines. Treatment of RMS cell lines with metabolic inhibitors, some of which are currently used in treating neuronal and inflammatory diseases, slows cell growth in vitro. In addition, many of the signature genes are co-expressed with HIF1α targets and are significantly upregulated in hypoxia culture condition, indicating a hypoxia-driven metabolic reprogramming in RMS. Hence, our central hypothesis is that the hypoxia-driven GABA metabolic pathway contributes to rhabdomyosarcoma malignancy and represents a novel therapeutic target (Figure. 1). This is based on the following two considerations: 1) the Hypoxia-GABA axis represents a mechanism of “metabolic checkpoint” that coordinates metabolic status with HIF1α-mediated oncogenic signaling, and in turn, determines the RMS malignancy; 2) a persistent metabolic rewiring renders RMS cells highly dependent on GABA metabolism (metabolic addiction), and the modulation of this process holds the promise of novel therapeutic interventions. To test these concepts, we will address the following questions: 1. Does hypoxia-driven metabolic rewiring in RMS confer a metabolic addiction on GABA? In silico reconstructions of such metabolic signature genes predicts both glutamine-glutamate and arginine-ornithine metabolic axis can give rise to GABA, which may serves as a anaplerotic substrate to replenish TCA cycle intermediates. We will coordinately assess the metabolo-transcriptome and systematically characterize the metabolic programs in RMS. By measuring the metabolic flux and metabolic gene expression in the context of modulating oxygen and HIF1α, we will determine whether hypoxia/HIF1α regulates GABA metabolic programs and, subsequently, will assess the contribution of glutamine and arginine to GABA flux. We will modulate GABA, glutamine and arginine metabolism through genetic approaches (Tet- ON inducible knockdown) and pharmacological approaches (metabolic inhibitors). As such, we will assess the impact of metabolic perturbation on RMS cell survival, proliferation, migration and differentiation in vitro. 2. Does GABA metabolism represent a novel therapeutic avenue in RMS? Our preliminary results indicate that metabolic inhibitors slow RMS cell growth in vitro. We will further assess the antitumor activity of GABA metabolism inhibitor in human RMS cell xenografts. Also, tumor samples will be processed to assess the level of metabolites in GABA metabolism under each treatment condition. Since several of these metabolic inhibitors are either clinically approved drugs or in various stages of preclinical/clinical studies, we expect that positive pre-clinical data will be able to be rapidly translated into clinical trials for treating RMS patients.