Abstract LB-195: Bortezomib sensitizes glioblastoma for NK cell immunotherapy
Authors: Navarro Andrea Gras , Rahman Aminur , Bahador Marzieh , Enger Martha Chekenya 
Published: July 2017

Introduction: Glioblastoma (GBM) is the most aggressive and lethal brain cancer in adults where best available therapy combining surgery and concomitant chemo radiation prolongs patient survival from 12.1 to 14.6 months. Approximately half of the patients benefit from addition of Temozolomide chemotherapy (those with methylated promoter of the DNA repair gene O6 methylguanine DNA methyltransferase). Immunotherapy has demonstrated unprecedented efficacy in solid tumours and kill targets in unique ways to the conventional therapies. Natural Killer (NK) cells are attractive effectors not only because of their ability to recognize and kill malignant and virus infected cells, but also their potential for modifying the tumor microenvironment to pro-inflammatory state. We have previously demonstrated NK-cell cytotoxicity against GBM. Here we hypothesize that misfolded protein response induced by pretreatment with proteasome inhibitors will sensitize GBM cells to NK cell therapy.

Methods: IC50 doses of Bortezomib, Carfilzomib and MG132 proteasome inhibitors were determined by viability assays in P3, 2012-018 and P22 patient-derived GBM cells, as well as in normal human astrocytes (NHA). Phenotyping, time and dose dependent cytotoxicity/apoptosis and degranulation after combination therapy with Bortezomib and NK cells from healthy donors was conducted with flow cytometry and western blotting used to confirm protein signaling.

Results: We demonstrated that Bortezomib is the most suitable proteasome inhibitor as it selectively killed GBM cells at low doses (IC50 39-42nM) while requiring higher doses (70nM) for NHA cells. In contrast, Carfilzomib and MG132 killed better the NHA than GBM cells. Bortezomib was more effective in combination treatment with NK cells, than monotherapy of Bortezomib or NK cells. The IC50 dose of Bortezomib was reduced by 40% from 42nM at 48hrs to 25nM at 24hrs when combined with effector:target (E:T) ratio 5:1 NK cells (p&lt0.05). E:T ratio of NK cells was also reduced by 75% in combination therapy, NK cell monotherapy killed only 45% of GBM cells at 20:1 ratio. The killing efficacy of combination treatment was not mediated by increased stress ligand expression, but Bortezomib attenuated autophagy and induced late apoptotic GBM cell death. Although Bortezomib did not increase degranulation or kill NK cells, importantly, it rather induced NK cell differentiation to mature CD56dimCD16-NKG2D+ subsets previously associated with increased natural cytotoxicity.

Conclusion: Bortezomib was the best proteasome inhibitor against GBM that synergistically enhanced NK cell killing. Current work is investigating the mechanisms regulating the augmented differentiation and NK cell cytotoxicity during the combination therapy in vitro and in vivo in mice.

Citation Format: Andrea Gras Navarro, Aminur Rahman, Marzieh Bahador, Martha Chekenya Enger. Bortezomib sensitizes glioblastoma for NK cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-195. doi:10.1158/1538-7445.AM2017-LB-195