Pretreatment of Glioblastoma with Bortezomib Potentiates Natural Killer Cell Cytotoxicity through TRAIL/DR5 Mediated Apoptosis and Prolongs Animal Survival

Abstract

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.

Sequential bortezomib and temozolomide treatment promotes immunological responses in glioblastoma patients with positive clinical outcomes: A phase 1B study

“…BTZ-induced tumour death generates immunogenic antigens and sensitizes to dendritic cell immunotherapy. 24,31,32 Thus, we investigated the patients' T cell activation and maturation status in blood and cytokines circulating in their plasma at baseline, after exposure to BTZ only (within the first 8 hours after administration), BTZ + TMZ (on days 4-7) and during the 3 weeks recovery period (days 11, 15, and 22) in cycle 1. We also investigated responses to stimulation with PMA/ionomycin or autologous tumour cells ex vivo of patients' PBMCs obtained at baseline (BTZ naïve) versus those obtained during recovery (day 22-56) after initial exposure to BTZ + TMZ.…”

“…Indeed, we demonstrated that natural killer (NK) cells treated with BTZ exhibited more mature, activated and cytotoxic CD57 + CD16 dim CD69 + phenotype, and that efficacy of combination treatment of GBM cells with BTZ + NK cells in vitro and in vivo was augmented by tumor necrosis factor related ligand (TRAIL)-receptor interactions, as well as tumor expression of stress-ligands recognized by activating NKG2D receptor. 24 Sequential combination of BTZ 48 hours before TMZ 164 mg/m 2 treatment, depleted MGMT mRNA in vivo, attenuated tumor growth and significantly prolonged animal survival. 22 Thus, this phase IB of our BORTEM-17 clinical trial (NCT03643549) was launched to investigate whether pretreatment of recurrent GBM patients with BTZ 1.3 mg/m 2 on days 1, 4, and 7, to deplete MGMT levels 48 hours before TMZ, commencing on days 3 to 7 every 4th week, might sensitize MGMT unmethylated GBM to TMZ chemotherapy.…”

Natural killer cells in patients with hematologic malignancies, solid tumors and in recipients of hematopoietic stem cell transplantation

“…GB stem-like cells are more susceptible to lysis by NK cells than differentiated GB cells [277]. In animal models, pretreatment of GB with bortezomib has been shown to promote NK cell cytotoxicity and to inhibit tumor growth ultimately leading to prolonged survival [278]. Varicella zoster virus (VZV) is the only virus with negative correlation with GB [169,279].…”

Inhibition of PLK4 might enhance the anti‐tumour effect of bortezomib on glioblastoma via PTEN/PI3K/AKT/mTOR signalling pathway

“…Previous studies have shown that bortezomib disrupts multiple downstream signalling pathways, such as the NF‐κB signalling pathway and ubiquitin‐proteasome pathway, and has an important role in the regulation of cell cycle, mitosis, cell viability, proliferation and apoptosis in glioblastoma cells . It also blocks autophagy flux mediated by the 26S proteasome, thereby inhibiting the elimination of damaged organelles, and inhibits the proliferation of glioblastoma. Polo‐like kinase 4 (PLK4) is critical for embryonic development and cell cycle control.…”