Background: Glioblastoma (GBM), a WHO Grade IV cancer, is the most common and most aggressive primary malignant brain tumor. Prognosis following best standard-of-care surgical resection, radiation and temozolomide (TMZ) chemotherapy is poor, with median overall survival 15-18 months. TMZ, which acts primarily by methylating DNA at O6-guanine, has marginal efficacy in ̃70% of patients due to tumor expression of O-6-methylguanine-DNA methyltransferase (MGMT) and there is no effective therapy at tumor recurrence. New treatment options are therefore desperately needed for GBM patients. LP-184 is an acylfulvene-derived prodrug that is activated by the oxidoreductase PTGR1 and primarily alkylates adenine at the N³-position. Bioinformatic analysis showed that PTGR1 is elevated in clinical GBM tumor samples compared to normal brain. LP-184 response is influenced by tumor DNA repair status, and acylfulvene-induced damage is mainly repaired by the transcription-coupled nucleotide-excision repair (TC-NER) pathway. Spironolactone, an FDA approved, blood-brain-barrier permeable diuretic, induces degradation of ERCC3 protein, a key TC-NER component. Response of NER-deficient GBM was tested through co-administration of LP-184 and spironolactone. Methods: Cell viability assays with exposure to LP-184, with and without spironolactone, were used to determine IC₅₀ values in vitro. Subcutaneous or intracranial tumor xenograft models were used to determine tumor volume or survival differences between control and LP-184 treated mice. Results: Nanomolar concentrations of LP-184 inhibited in vitro viability of multiple GBM cell models including 4 NCI-60 GBM cell lines (mean IC₅₀= 286 nM), the TMZ-resistant LN-18 cell line (IC₅₀= 46 nM), M1123 GBM neurosphere line (IC₅₀= 151 nM), and a low passage GBM neurosphere isolate (IC₅₀= 306 nM). Co-treatment with spironolactone resulted in a 3-6 fold decrease in LP-184 IC₅₀ values in M1123, Mayo39, and U87 cultures. Pharmacokinetic analyses in SCID mice following a single LP-184 infusion (4 mg/kg, i.v.) showed favorable CNS bioavailability with brain/plasma ratio 0.11 (C_max= 839 nM) and brain tumor/plasma ratio 0.2 (C_max= 2.53 µM). LP-184 (4mg/kg i.v., q.o.d X 4) induced complete regression of all pre-established s.c. U87 xenografts (N = 5) and 3 of 6 M1123 s.c. xenografts (mean tumor volumes: 1456 ± 349 mm 3 controls vs 84 ± 39 LP-184, p < 0.001) and statistically significantly prolonged survival of mice bearing pre-established orthotopic U87 and M1123 xenografts (p < 0.0001). Conclusions: These preclinical results establish LP-184 as a promising new agent for future clinical testing in patients with GBM and suggest that LP-184 may be particularly efficacious in patients with intrinsic or spironolactone-induced NER deficient tumors.