Abstract Background: Glioblastoma with unmethylated O6-methyl guanine DNA methyltransferase (MGMT) promoter is highly resistant to temozolomide (TMZ) chemotherapy. Strategies that ameliorate drug resistance are sorely needed. Recent trials of the proteasome inhibitor bortezomib (BTZ) (Velcade) in combination with various drugs failed due to inappropriate schedule timing and dosing. We hypothesized that pretreatment with BTZ prior to TMZ administration may sensitize glioblastoma cell to TMZ chemotherapy. Methods: We investigated treatment efficacy through DNA damage, apoptosis and autophagy flux by flow cytometry, western blotting, long-lived protein degradation assays, electron and fluorescence microscopy in cell lines (U87, T98G, HF66) and patient biopsy-derived cells (P3, 2012-18, BG5 and BG7). Treatment efficacy and tolerability was investigated in vivo in mice implanted orthotopically with patient-derived GBM xenografts and subsequently treated with human equivalent dose (HED) of BTZ 1.3 mg/m2 on days 1, 4, 8 and 11 for two cycles during TMZ 82 mg/m2 or 164 mg/m2 by oral gavage 5 days/week for 5 weeks. MRI, mouse survival times, tandem LC-MS/MS and clinical chemistry were used to monitor tumor growth and evaluate tissue and blood samples for biomarkers of treatment efficacy and toxicity. Results: Patient-derived glioblastoma cells were universally more sensitive to BTZ than carfilzomib or MG-132 (P<0.0001), while their sensitivity to TMZ was strongly associated with MGMT promoter methylation status (P<0.0001). BTZ depleted MGMT protein (P<0.001) and mRNA (P<0.0001) in TMZ resistant tumor cells and sensitized to chemotherapy through induction of prominent DNA damage, G2-S phase cell cycle arrest and apoptosis with half the IC50 doses for both drugs. BTZ simultaneously abrogated TMZ-induced autophagic flux indicated by p62 accumulation, inhibition of utophagosome fusion and degradation of long-lived proteins. Human equivalent doses of BTZ 1.3 mg/m2 and TMZ 164 mg/m2 prolonged progression free and overall survival (P<0.0001), through diminished tumor growth (P<0.05), angiogenesis (P<0.01) and proliferation (P<0.05). Loss of structural and catalytic proteasomal subunits confirmed target inhibition at the therapeutically effective dose (P<0.01). Peroxiredoxin, thioredoxin and catalase antioxidant enzymes (P<0.01), alanine aminotransferase (P<0.05) and MGMT mRNA expression (P<0.01) were downregulated in combination treated animals, serving as accessible biomarkers of response. Conclusion: Pretreatment with BTZ chemo-sensitized resistant glioblastoma through aborted autophagic flux and MGMT depletion to augment DNA damage, apoptosis and prolonged survival. Our findings warrant a controlled investigation of the treatment schedule in selected patients. Citation Format: Mohummad A. Rahman, Andrea G. Navarro, Jorunn Brekke, Christian Bindesbøll, Agnete Engelsen, Shahin Sarowar, Marzieh Bahador, Bjørn T. Gjertsen, Dorota Goplen, Per Ø. Enger, Frode Selheim, Anne Simonsen, Martha Chekenya. Bortezomib sensitizes glioblastoma with unmethylated MGMT promoter to temozolomide-chemotherapy through MGMT depletion and abrogated autophagy flux [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2928.