Proteome Responses to Acute Inhibition of De Novo Sphingolipid Synthesis Suggest Cancer Combination Therapies

Background: Sphingolipids are essential structural and signaling lipids that support membrane integrity and govern cell fate decisions. While the consequences of chronic sphingolipid inhibition have been extensively explored, the immediate cellular responses to acute suppression of sphingolipid synthesis remain poorly defined. Methods: We analyzed subcellular proteomic changes following an acute reduction in sphingolipid levels induced by myriocin, an inhibitor of de novo sphingolipid synthesis. We then evaluated the cytotoxicity of co-treatment with myriocin and inhibitors of the altered pathways in cancer cells. Results: We found that de novo sphingolipid synthesis is sensitive to myriocin, an inhibitor of serine palmitoyltransferase (SPT), and can be efficiently inhibited within 4 h of treatment. Cells respond to reduced sphingolipid levels by rapidly remodeling their proteome. Mass spectrometry analysis revealed changes in the abundance of hundreds of proteins across the membrane, cytosolic, and nuclear fractions. Gene set enrichment analysis revealed alterations in the proteome across several pathways involved in protein and lipid homeostasis and stress responses, including upregulation of cholesterol homeostasis and lysosome. Co-treatment with myriocin and cholesterol synthesis or lysosomal function inhibitors synergistically reduced cancer cell viability by promoting apoptosis rather than other forms of programmed cell death. Conclusions: Together, our work provides insights into how cells rapidly rewire the abundance of certain protein classes in response to reduced sphingolipid levels and identifies signaling and metabolic pathways that can be exploited for therapeutic intervention.