Abstract Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder for which there is currently no treatment or cure. Affected individuals have difficulty accomplishing everyday tasks such as lifting one’s arms above their head, impacting quality of life. Much research has been done trying to elucidate the mechanism of disease pathology but there has been little congruence apart from apoptosis via DUX4, the toxic protein associated with FSHD. We took advantage of this phenotype to design a genome-wide complimentary CRISPR-Cas9 loss of function and activation screens for genetic modifiers of DUX4 toxicity. These screens identified hypoxia signaling as a novel pathway relating to FSHD pathology with therapeutic potential. Small molecule inhibitors that target modifiers of this pathway, including the FDA-approved compound everolimus, reduced DUX4-mediated cell death and FSHD biomarkers, demonstrating the viability of targeting hypoxia signaling as a potential therapy. In this project we intend to further explore the mechanism of how hypoxia signaling relates to DUX4-mediated pathology (Specific aims 1). The PI3K/Akt/mTOR signaling axis that interacts with hypoxia signaling will be modulated through knock-down and/or pharmacological inhibition for their effect on DUX4-mediated apoptosis. The consequence of autophagy modulation through the mTOR inhibitor everolimus will be explored. The relationship between DUX4-induced hypoxia and metabolic dysfunction will be assessed through RNAseq, Seahorse assays of oxygen consumption and extracellular acidification rates, modulation of oxidative phosphorylation and reactive oxygen species generation. We will additionally test the therapeutic potential of everolimus in a DUX4-inducible mouse model of FSHD (Specific aims 2). We will measure effects on muscle function through treadmill endurance, grip strength and ex vivo contractile force. Using microscopy, we will examine effects on muscle histology and markers of hypoxic signaling while exploring if this pathology is affected by the metabolic profile of the individual muscles. Lastly, effect on FSHD biomarkers will be assessed both in the mouse model and validated using patient biopsy myocytes. If successful, this project will elucidate a new mechanism of FSHD pathology for therapeutic targeting and identify everolimus as a therapeutically promising compound whose FDA status allows for a shorter timeframe before patient availability.