Müge Sak1,2, Cory T Zumbar1, Paul D King3, Xiaohui Li3, Caroline S Mifsud3, Aisulu Usubalieva3, Charles D Anderson1, Hailey M Chesnick3, Joseph P McElroy4, Arnab Chakravarti5, Eric C Burton6, Norman L Lehman7,8,9. 1. Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY, 40202, USA. 2. Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, 40202, USA. 3. The Department of Pathology, Ohio State University, Columbus, OH, 43212, USA. 4. Department of Biomedical Informatics, Ohio State University, Columbus, OH, 43212, USA. 5. Department of Radiation Oncology, Ohio State University, Columbus, OH, 43212, USA. 6. Neuro-Oncology Branch, National Cancer Institute, Bethesda, MD, 20892, USA. 7. Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY, 40202, USA. nllehman1@gmail.com. 8. Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, 40202, USA. nllehman1@gmail.com. 9. The Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA. nllehman1@gmail.com.
Abstract
INTRODUCTION: Glioblastoma remains difficult to treat and patients whose tumors express high levels of O6-methylguanine DNA methyltransferase (MGMT) usually respond poorly to standard temozolomide chemotherapy. We have previously shown that the selective AURKA inhibitor alisertib potently inhibits growth of glioblastoma cells. METHODS: We used colony formation assays, annexin V binding, and western blotting to examine the effects of alisertib on the antiproliferative capabilities of carboplatin and irinotecan in glioblastoma cells. RESULTS: In colony formation assays, alisertib potentiated the antiproliferative effects of both carboplatin and irinotecan, often synergistically, including against glioblastoma tumor stem-like cells, as demonstrated by Chou-Talalay and Bliss statistical analyses. Western blotting showed that high MGMT expression in cell lines correlated with more pronounced potentiation of carboplatin's growth inhibitory effects by alisertib, while low MGMT expression correlated with stronger potentiation of irinotecan by alisertib. This pattern was also observed when these drug combinations were tested for their ability to induce apoptosis via annexin V binding assays. MGMT knockdown increased apoptosis caused by combined alisertib and irinotecan, while exogenous MGMT overexpression increased apoptosis from alisertib and carboplatin combination treatment. CONCLUSIONS: These results suggest that tumor MGMT expression levels may be predictive of patient response to these drug combinations, and importantly that the combination of alisertib and carboplatin may be selectively effective in glioblastoma patients with high tumor MGMT who are resistant to standard therapy. Since clinical experience with alisertib, carboplatin and irinotecan as single agents already exists, these findings may provide rationale for the design of clinical trials for their use in combination treatment regimens.
INTRODUCTION:Glioblastoma remains difficult to treat and patients whose tumors express high levels of O6-methylguanine DNA methyltransferase (MGMT) usually respond poorly to standard temozolomide chemotherapy. We have previously shown that the selective AURKA inhibitor alisertib potently inhibits growth of glioblastoma cells. METHODS: We used colony formation assays, annexin V binding, and western blotting to examine the effects of alisertib on the antiproliferative capabilities of carboplatin and irinotecan in glioblastoma cells. RESULTS: In colony formation assays, alisertib potentiated the antiproliferative effects of both carboplatin and irinotecan, often synergistically, including against glioblastoma tumor stem-like cells, as demonstrated by Chou-Talalay and Bliss statistical analyses. Western blotting showed that high MGMT expression in cell lines correlated with more pronounced potentiation of carboplatin's growth inhibitory effects by alisertib, while low MGMT expression correlated with stronger potentiation of irinotecan by alisertib. This pattern was also observed when these drug combinations were tested for their ability to induce apoptosis via annexin V binding assays. MGMT knockdown increased apoptosis caused by combined alisertib and irinotecan, while exogenous MGMT overexpression increased apoptosis from alisertib and carboplatin combination treatment. CONCLUSIONS: These results suggest that tumorMGMT expression levels may be predictive of patient response to these drug combinations, and importantly that the combination of alisertib and carboplatin may be selectively effective in glioblastomapatients with high tumorMGMT who are resistant to standard therapy. Since clinical experience with alisertib, carboplatin and irinotecan as single agents already exists, these findings may provide rationale for the design of clinical trials for their use in combination treatment regimens.
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