Exploring the role of hypoxia signaling and its inhibition as a therapy for Facioscapulohumeral muscular dystrophy

摘要 面肩肱型肌营养不良症（FSHD）是一种神经肌肉疾病， 治疗或治愈。受影响的人难以完成日常任务，如举起手臂 影响生活质量。许多研究试图阐明 但是除了通过毒性蛋白DUX 4引起的细胞凋亡外， 与FSHD有关。我们利用这种表型设计了一个全基因组互补的 CRISPR-Cas9功能丧失和激活筛选DUX 4毒性的遗传修饰剂。这些屏幕 鉴定了缺氧信号传导作为与具有治疗潜力的FSHD病理学相关的新途径。小 靶向该途径修饰剂的分子抑制剂，包括FDA批准的化合物依维莫司， 减少DUX 4介导的细胞死亡和FSHD生物标志物，证明靶向缺氧的可行性 信号作为一种潜在的疗法。本项目拟进一步探讨缺氧如何影响 信号传导与DUX 4介导的病理学有关（具体目的1）。PI 3 K/Akt/mTOR信号轴， 与缺氧信号传导的相互作用将通过敲低和/或药理学抑制来调节， 它们对DUX 4介导的细胞凋亡的影响。通过mTOR调节自噬的结果 将探索抑制剂依维莫司。DUX 4诱导的缺氧与代谢的关系 功能障碍将通过RNAseq、耗氧量和细胞外的海马测定来评估。 酸化速率，氧化磷酸化和活性氧生成的调节。我们将 此外，还测试了依维莫司在DUX 4诱导的FSHD小鼠模型中的治疗潜力（特异性 目标2）。我们将通过跑步机耐力、握力和体外试验来测量对肌肉功能的影响。 收缩力使用显微镜，我们将检查对肌肉组织学和缺氧标志物的影响。 信号，同时探索这种病理是否受到个体肌肉代谢特征的影响。最后， 将在小鼠模型中评估对FSHD生物标志物的影响，并使用患者活检进行验证 肌细胞如果成功，该项目将阐明FSHD病理学的新机制， 靶向并鉴定依维莫司作为治疗上有前景的化合物，其FDA状态允许 缩短患者可用时间。

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.