Abstract
Overexpression of O6-methylguanine DNA methyltransferase (MGMT) contributes to resistance to chemo-radiation therapy (CRT) in brain tumors. We previously demonstrated that non-ablative radiation improved delivery of anti-MGMT morpholino oligonucleotides (AMONs) to reduce MGMT levels in subcutaneous tumor xenografts. We evaluate this approach to enhance CRT efficacy in rat brain tumor xenograft models. The impact of radiation on targeted delivery was evaluated using fluorescent oligonucleotides (f-ON). In vitro, f-ON was localized to clathrin-coated vesicles, endosomes, and lysosomes using confocal microscopy in T98G glioma cells. In vivo, fluorescence was detected in pre-radiated, but not non-radiated Long Evans (non-tumor bearing) rat brains. Cranial radiation (2 Gy) followed by AMONs (intravenous, 10.5 mg/kg) reduced MGMT expression by 50% in both orthotopic cerebellar D283 medulloblastoma and intracerebral H460 non-small cell lung carcinoma (NSCLC) xenograft models. To evaluate the efficacy, AMONs concurrent with CRT (2 Gy radiation plus oral 20 mg/kg temozolomide ×4 days) reduced tumor volumes in the medulloblastoma model (p = 0.012), and a similar trend was found in the NSCLC brain metastasis model. We provide proof of concept for the use of non-ablative radiation to guide and enhance the delivery of morpholino oligonucleotides into brain tumor xenograft models to reduce MGMT levels and improve CRT efficacy.
Highlights
These authors contributed : Prakash Ambady, Yingjen Jeffrey Wu
We have previously reported the successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-methylguanine DNA methyltransferase (MGMT) morpholino oligonucleotides (AMONs) sequences to block MGMT mRNA translation [10]
Compared to traditional chemo-radiation therapy (CRT) (2 Gy + temozolomide), we demonstrated the addition of AMONs (15 μM) at 24 h after 2 Gy radiation, significantly reduced D283 medulloblastoma cell viability (Supplemental Fig. 2A), and increase the presence of pro-apoptotic cleaved PARP protein (Supplemental Fig. 2B)
Summary
Morpholino antisense oligonucleotides are short synthetic nucleotide sequences that bind to complementary messenger RNA and block protein synthesis They are specific, water-soluble, non-toxic, uncharged, and extremely stable under physiologic conditions, and are not cleaved by endogenous nucleases, providing an excellent platform for potential targeted therapies in oncology [7,8,9]. We have previously reported the successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotides (AMONs) sequences to block MGMT mRNA translation [10]. We evaluate the use of clinically relevant non-ablative doses of radiation to guide and enhance the delivery of intravenously administered AMONs to brain tumor sites to silence MGMT and enhance the efficacy of CRT in orthotopic rat models of MGMT-positive human brain tumors. Targeted silencing of MGMT in pre-radiated tumor sites using our novel approach, without measurable MGMT silencing in nonradiated sites, may limit systemic toxicities associated with prior approaches to deplete MGMT in combination with temozolomide [18,19,20]
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