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

摘要 面肩肱骨肌营养不良症(FSHD)是一种神经肌肉疾病，目前尚无治疗方法 治疗或治愈。受影响的人难以完成日常任务，如举起手臂 在他们头顶上，影响着生活质量。人们已经做了大量的研究，试图阐明 疾病病理，但除了通过有毒蛋白质DUX4进行的细胞凋亡外，几乎没有什么一致的 与FSHD相关。我们利用这种表型设计了一种全基因组的免费 CRISPR-Cas9功能丧失和DUX4毒性遗传修饰物的激活筛选。这些屏幕 低氧信号被认为是与FSHD病理相关的具有治疗潜力的新途径。小的 靶向这一途径的修饰物的分子抑制剂，包括FDA批准的化合物伊波利莫斯， 减少DUX4介导的细胞死亡和FSHD生物标志物，证明靶向缺氧的可行性 作为一种潜在的治疗方法。在这个项目中，我们打算进一步探索缺氧的机制 信号转导与DUX4介导的病理有关(特异性目标1)。PI3K/Akt/mTOR信号轴 与低氧信号的相互作用将通过击倒和/或药物抑制来调节 它们对DUX4介导的细胞凋亡的影响。通过mTOR进行自噬调控的后果 将探索伊波利莫斯的抑制剂。DUX4诱导的低氧与代谢的关系 功能障碍将通过RNAseq、海马氏氧气消耗和细胞外试验进行评估 酸化速率、氧化磷酸化的调节和活性氧物种的产生。我们会 此外，在DUX4诱导的FSHD(特异性)小鼠模型中测试伊波利莫斯的治疗潜力 目标2)。我们将通过跑步机耐力、握力和体外实验来衡量对肌肉功能的影响 收缩力。在显微镜下，我们将检查肌肉组织学和低氧标志物的影响 在探索这种病理是否受到个别肌肉代谢状况的影响时发出信号。最后， 对FSHD生物标志物的影响将在小鼠模型中进行评估，并通过患者活检进行验证 肌细胞。如果成功，该项目将阐明FSHD病理治疗的新机制。 靶向并确定依维莫司是一种有治疗前景的化合物，其FDA状态允许 缩短患者出院前的时间。

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.