Defective autophagy in GNE myopathy is rescued by inhibition of noncanonical Akt–mTORC1 activation across multiple isogenic models

Abstract GNE myopathy is a recessive autosomal disease caused by mutations in glucosamine (UDP- N -acetyl)-2-epimerase/ N -acetylmannosamine kinase (GNE), characterized by impaired sialic acid biosynthesis and the formation of rimmed vacuoles. Similar to other autophagic vacuolar myopathies, defective autophagy has been implicated in disease pathogenesis; however, the underlying molecular mechanisms remain poorly understood. By performing transcriptome analysis on two independent GNE myoblast models derived from human pluripotent stem cells, we identified multiple autophagy-related gene sets as pathogenic signatures of GNE myopathy. These predictions were biochemically validated using Gne -knockout C2C12 myoblasts. Mechanistically, our data reveal that aberrant activation of the noncanonical AKT–mTORC1 pathway—driven by excessive extracellular matrix production—induces inhibitory phosphorylation of ULK1, thereby suppressing autophagy initiation. To identify therapeutic targets, we performed a transcriptome-based drug screen using gene signature reversal, which nominated copanlisib, an FDA-approved Pi3k inhibitor, as a promising candidate. Functional validation in human pluripotent stem cell-derived neuromuscular organoids demonstrated that copanlisib reactivates autophagy via restoration of ULK1 activity. Together, our findings uncover a mechanistic link between extracellular matrix dysregulation and impaired autophagy in GNE myopathy and highlight copanlisib as a potential therapeutic strategy.