BCL2L13 Influences Autophagy and Ceramide Metabolism without Affecting Temozolomide Resistance in Glioblastoma

Abstract Temozolomide (TMZ) resistance in glioblastoma (GBM) arises through metabolic rewiring that links mitochondrial function, autophagy balance, and sphingolipid metabolism. TMZ-resistant (R) U251 cells exhibited suppressed apoptosis and complete blockade of autophagy flux, evidenced by MAP1LC3/LC3-II and SQSTM1/p62 accumulation and insensitivity to bafilomycin A 1 . BCL2L13, strongly upregulated in R cells, emerged as a dual regulator of mitophagy and ceramide metabolism. BCL2L13 knockdown (KD) produced opposite effects in TMZ-sensitive (NR) and resistant cells: in NR cells, KD elevated LC3-II, reduced respiratory reserve, and triggered compensatory lipid synthesis; in R cells, KD decreased LC3-II without restoring flux or TMZ sensitivity. Lipidomic profiling revealed that BCL2L13 loss reactivated CERS6 in NR cells, increasing C16:0 and mid-chain ceramides, while relieving CERS2 inhibition in R cells, elevating very-long-chain (C22–C24) and glycosylated ceramides. These distinct sphingolipid signatures were confirmed by PLS-DA and KEGG enrichment, which highlighted steroid hormone, arachidonic, and linoleic acid metabolism in NR KD cells versus neuroactive ligand-receptor and signaling pathways in R KD cells. Together, these findings position BCL2L13 as a molecular integrator of mitochondrial respiration, autophagy flux, and CERS-dependent lipid remodeling, unveiling a context-specific metabolic mechanism that supports GBM cell survival under conditions of chemotherapeutic stress.