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bioRxiv · 10.64898/2026.03.19.712341

A treatment-resilient lipid metabolic program drives temozolomide resistance in glioblastoma

Naghibzadeh, K.·Barzegar Behrooz, A.·Kavoosi, M.·Cordani, M.·Los, M. J.·Pecic, S.·Vitorino, R.·Vitorino, C.·Ravandi, A.·Shojaei, S.·Ghavami, S.
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Abstract

Temozolomide (TMZ) resistance remains a major barrier to durable control of glioblastoma (GB). Our previous studies showed that simvastatin can enhance TMZ-induced cell death in non-resistant GB cells by disrupting autophagosome-lysosome fusion and engaging stress-response pathways, whereas established TMZ-resistant cells maintain impaired autophagy flux and remain refractory to TMZ, simvastatin, and their combination. Here, we asked whether this loss of therapeutic responsiveness is accompanied by a treatment-resilient lipid state. Targeted LC-MS quantified 304 lipid species across 25 analytical classes in non-resistant (NR) and TMZ-resistant (R) U251-mKate cells under control, simvastatin (ST), TMZ, and TMZ-ST conditions. Global heatmap, principal-component, volcano, and exploratory PLS-DA analyses demonstrated persistent NR/R lipidomic separation across all four treatment states. Resistant cells recurrently displayed enrichment of lysophospholipids, selected sphingolipids/glycosphingolipids, and cholesteryl esters, with depletion or redistribution of several glycerophospholipid and diacylglycerol pools. A family-resolved analysis across 19 lipid families showed that resistance status was most strongly associated with phosphatidylinositol (PI; PERMANOVA R^2=0.52), phosphatidylglycerol (PG; R^2=0.49), lysophosphatidylcholine (LPC; R^2=0.46), lysophosphatidylethanolamine (LPE; R^2=0.45), ether/plasmalogen phosphatidylcholine (R^2=0.45), and phosphatidylcholine (R^2=0.41). Structure-informed target prediction of discriminant lipids generated pathway hypotheses involving Rap1, PI3K-Akt, phospholipase D, calcium, PPAR, and lipid-metabolic signaling. Transmission electron microscopy showed persistent vesicle-rich, autophagosome-like architecture in resistant cells across treatment conditions, consistent with the previously established late-stage autophagy defect. These data extend the autophagy-cholesterol model of TMZ resistance to a broader membrane-remodeling program and indicate that failure of statin sensitization is associated with coordinated lipid-family remodeling rather than a single lipid species or pathway. The identified lipid families and cholesterol-storage phenotype provide testable vulnerabilities for future functional validation. An exploratory family-level linear SVM analysis provided an orthogonal proof-of-concept: several membrane and storage-lipid families retained complete NR/R separation when an entire treatment was withheld, but these internal results were interpreted as supplementary evidence rather than as a validated classifier.

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Naghibzadeh, K., Barzegar Behrooz, A., Kavoosi, M., Cordani, M., Los, M. J., Pecic, S., Vitorino, R., Vitorino, C., Ravandi, A., Shojaei, S., Ghavami, S.. 2026-03-21. A treatment-resilient lipid metabolic program drives temozolomide resistance in glioblastoma. https://doi.org/10.64898/2026.03.19.712341

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