Abstract
The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is epigenetically silenced in some tumors by MGMT gene promoter methylation. MGMT-hypermethylated solid tumors have enhanced susceptibility to the cytotoxic effects of alkylating chemotherapy such as temozolomide, compared with non-methylated tumors. In glioblastoma, subjects with MGMT hypermethylation have significantly longer survival rates after chemoradiotherapy. We report the first successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotide (AMON) sequences. We demonstrate >40% reduction in the in vitro proliferation index and cell viability in radiation-primed MGMT-expressing human solid tumor cells treated with a single dose of AMONs and temozolomide. We further demonstrate the feasibility of using a non-ablative dose of radiation in vivo to guide and enhance the delivery of intravenously administered AMONs to achieve 50% MGMT knockdown only at radiation-primed tumor sites in a subcutaneous tumor model. Local upregulation of physiological endocytosis after radiation may have a role in radiation-guided uptake of AMONs. This approach holds direct translational significance in glioblastoma and brain metastases where radiation is part of the standard of care; our approach to silence MGMT could overcome the significant problem of MGMT-mediated chemoresistance.
Highlights
Glioblastoma is an aggressive primary brain tumor that carries a poor prognosis even after aggressive resection and standard-ofcare chemoradiotherapy (CRT)
Expression of methylguanine DNA methyltransferase (MGMT) was verified in multiple cancer cell types and three MGMT-expressing solid tumor cell lines (T98G glioma, and H460 and A549 non-small cell lung carcinoma (NSCLC)) were selected for further experiments
Of the 5 different time points we investigated, the best MGMT knockdown (87.4 ± 5.3%) was achieved when cells were exposed to anti-MGMT morpholino oligonucleotide (AMON) 24 h after radiation compared with nonradiated control (Figure 1b)
Summary
Glioblastoma is an aggressive primary brain tumor that carries a poor prognosis even after aggressive resection and standard-ofcare chemoradiotherapy (CRT). The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is a suicide enzyme that unmethylates these sites to restore genome stability.[2,3,4,5,6,7,8] Via this mechanism, MGMT enzyme expression has a key role in resistance to CRT.[2,3,5,6] The MGMT gene is epigenetically silenced in about 45% of glioblastoma cases by methylation of CpG islands, predominantly located in the 5′ gene promoter region.[9] Tumor cells with MGMT gene promoter methylation have enhanced susceptibility to the cytotoxic effects of radiation and/or alkylating agents such as temozolomide due to their inability to repair the CRT-mediated DNA damage. This correlates with a significant survival benefit in glioblastoma patients with MGMT promoter methylation who receive
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