BackgroundResistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ.MethodsCell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan–Meier method.ResultsPre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood–brain barrier, diminished proteasome activity and significantly prolonged animal survival.ConclusionBTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

Melanoma patients carry a high risk of developing brain metastases, and improvements in survival are still measured in weeks or months. Durable disease control within the brain is impeded by poor drug penetration across the blood-brain barrier, as well as intrinsic and acquired drug resistance. Augmented mitochondrial respiration is a key resistance mechanism in BRAF -mutant melanomas but, as we show in this study, this dependence on mitochondrial respiration may also be exploited therapeutically. We first used high-throughput pharmacogenomic profiling to identify potentially repurposable compounds against BRAF -mutant melanoma brain metastases. One of the compounds identified was β-sitosterol, a well-tolerated and brain-penetrable phytosterol. Here we show that β-sitosterol attenuates melanoma cell growth in vitro and also inhibits brain metastasis formation in vivo. Functional analyses indicated that the therapeutic potential of β-sitosterol was linked to mitochondrial interference. Mechanistically, β-sitosterol effectively reduced mitochondrial respiratory capacity, mediated by an inhibition of mitochondrial complex I. The net result of this action was increased oxidative stress that led to apoptosis. This effect was only seen in tumor cells, and not in normal cells. Large-scale analyses of human melanoma brain metastases indicated a significant role of mitochondrial complex I compared to brain metastases from other cancers. Finally, we observed completely abrogated BRAF inhibitor resistance when vemurafenib was combined with either β-sitosterol or a functional knockdown of mitochondrial complex I. In conclusion, based on its favorable tolerability, excellent brain bioavailability, and capacity to inhibit mitochondrial respiration, β-sitosterol represents a promising adjuvant to BRAF inhibitor therapy in patients with, or at risk for, melanoma brain metastases. Electronic supplementary material The online version of this article (10.1186/s40478-019-0712-8) contains supplementary material, which is available to authorized users.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC–MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24–46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33–76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252–0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295–0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood–brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284–0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

Human cytomegalovirus (HCMV) antigens in glioblastoma (GBM) present opportunities for personalised immunotherapy. However, their presence in GBM tissue is still under debate, and evidence of their impact on functional immune responses and prognosis is sparse. Here, we investigated the presence of pp65 (UL83) and immediate early 1 (IE-1) HCMV antigens in a cohort of Norwegian GBM patients (n = 177), using qPCR, immunohistochemistry, and serology. HCMV status was then used to investigate whether viral antigens influenced immune cell phenotype, infiltration, activation and patient survival. Pp65 and IE-1 were detected by qPCR in 23% and 43% of GBM patients, respectively. Furthermore, there was increased seropositivity in GBM patients relative to donors (79% vs. 48%, respectively; Logistic regression, OR = 4.05, 95%CI [1.807-9.114], P = 0.001, also when adjusted for age (OR = 2.84, 95%CI [1.110-7.275], P = 0.029). Tissue IE-1-positivity correlated with increased CD3+CD8+ T-cell infiltration (P < 0.0001), where CD8+ effector memory T (TEM) cells accounted for the majority of CD8+T cells compared with peripheral blood of HCMV+ patients (P < 0.0001), and HCMV+ (P < 0.001) and HCMV− (P < 0.001) donors. HLA-A2/B8-restricted HCMV-specific CD8+ T cells were more frequent in blood and tumor of HCMV+ GBM patients compared with seronegative patients, and donors irrespective of their serostatus. In biopsies, the HCMV-specific CD8+ TEM cells highly expressed CTLA-4 and PD-1 immune checkpoint protein markers compared with populations in peripheral blood (P < 0.001 and P < 0.0001), which expressed 3-fold greater levels of CD28 (P < 0.001 and P < 0.0001). These peripheral blood T cells correspondingly secreted higher levels of IFNγ in response to pp65 and IE-1 peptide stimulation (P < 0.001). Thus, despite apparent increased immunogenicity of HCMV compared with tumor antigens, the T cells were tolerised, and HCMV status did not impact patient survival (Log Rank3.53 HR = 0.85 95%CI [0.564-1.290], P = 0.45). Enhancing immune functionality in the tumor microenvironment thus may improve patient outcome.

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&lt;0.0001), while their sensitivity to TMZ was strongly associated with MGMT promoter methylation status (P&lt;0.0001). BTZ depleted MGMT protein (P&lt;0.001) and mRNA (P&lt;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&lt;0.0001), through diminished tumor growth (P&lt;0.05), angiogenesis (P&lt;0.01) and proliferation (P&lt;0.05). Loss of structural and catalytic proteasomal subunits confirmed target inhibition at the therapeutically effective dose (P&lt;0.01). Peroxiredoxin, thioredoxin and catalase antioxidant enzymes (P&lt;0.01), alanine aminotransferase (P&lt;0.05) and MGMT mRNA expression (P&lt;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.