Neuromuscular Organoids Recapitulate Defective Autophagy in GNE Myopathy and Highlight Therapeutic Rescue by PI3K Inhibition

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 (hPSCs), we identified multiple autophagy-related gene sets as pathogenic signatures of GNE myopathy. These predictions were biochemically validated using Gne-knockout (KO) C2C12 myoblasts. Mechanistically, our data reveal that aberrant activation of the non-canonical 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 hPSC-derived neuromuscular organoids demonstrated that copanlisib reactivates autophagy via restoration of ULK1 activity. Together, our findings uncover a mechanistic link between ECM dysregulation and impaired autophagy in GNE myopathy and highlight copanlisib as a potential therapeutic strategy.