Effect of ATP7A knockdown on gene expression in glioma stem cells

Copper-related cell death, cuproptosis, involves accumulation of intracellular copper that triggers mitochondrial lipoylated protein aggregation and destabilization of iron–sulfur cluster proteins. Here, glioblastoma (GBM) stem cells (GSCs) displayed resistance to cuproptosis with circadian variation of intracellular copper levels, unlike differentiated GBM cells (DGCs) or neural stem cells (NSCs). CRISPR screening of copper regulators under concurrent treatment with copper ionophore or clock disruption revealed dependency on ATPase Copper Transporting Alpha (ATP7A). Circadian control of copper homeostasis was mediated by core clock transcription factor, Basic helix-loop-helix ARNT-like protein 1 (BMAL1). In turn, ATP7A promoted tumor cell growth through regulation of fatty acid desaturation. Copper levels negatively fed back onto the circadian clock through SQSTM1 (sequestosome 1/p62)-mediated lysosomal degradation of BMAL1. Targeting cuproptosis and circadian clock or fatty acid desaturation generated synergistic anti-tumor effects. Thus, crosstalk between the circadian rhythm and copper sustains GSCs, reshaping fatty acid metabolism and promoting drug resistance.