Abstract 5087: Radiation enhances the intracellular delivery of anti-MGMT morpholino oligonucleotides (AMONs) to enhance the cytotoxicity of chemo-radiation in cancer cells
Authors: Ambady Prakash , Wu Jeffrey , Kersch Cymon , Morris DreeAnna , Pagel Michael , Muldoon Leslie L. , Neuwelt Edward 
Published: July 2017

Background: This study evaluates the use of a sub-lethal dose of ionizing radiation to guide and enhance the intracellular delivery of morpholino antisense nucleotides designed to block O6-methylguanine DNA methyltransferase (MGMT) to enhance the cytotoxicity of chemo-radiation therapy (CRT). MGMT is a DNA repair enzyme and its expression is a well-known mechanism for resistance to CRT in glioblastoma. Epigenetic silencing of this enzyme by promotor methylation is associated with better response and clinically relevant survival benefits after CRT. We designed anti-MGMT morpholino oligonucleotides (AMON) using the stable vivo-morphalinos backbone. Intracellular delivery of morpholinos, particularly in a manner that could be clinically feasible, continues to be a major challenge. We use sublethal ionizing radiation to enhance the delivery of AMON to transiently reduced MGMT protein expression. We previously demonstrated that sublethal ionizing radiation enhances the delivery of AMONs to reduce MGMT expression in human cancer cells (T98G glioma and H460 and A549 non-small cell lung cancer cell lines) in vitro as well as the targeted knockdown of MGMT with systemically administered AMONsin pre-irradiated tumors. We now test whether blocking MGMT will enhance the cytotoxicity of CRT, and investigate the mechanism by which radiation permits cellular uptake of AMONs.

Methods: MGMT expressing, human T98G glioblastoma cells were used for all in-vitro studies. AMONs and non-specific fluorescent tagged control morpholinos were synthesized and purchased from Gene Tools, LLC (Philomath, OR, USA). High-resolution widefield microscopy with image deconvolution was performed to confirm the intracellular delivery. Cells were exposed to 1 Gy radiation; 24 hrs post radiation cells were treated with AMONs. Cells were then exposed to temozolomide (75 or 150 μg/mL) on day 3. Lysates were obtained on days 4 and 7. Expression of cellular MGMT protein, apoptosis markers, and key endocytosis proteins were characterized using immunoblotting.

Results: Our results demonstrate that MGMT silencing using a single dose of AMONs delivered with sublethal dose of radiation increased apoptosis by 50% and reduces cell viability by 50% at one and two days after therapy. Further, we demonstrate an increase in caveolin 1, clathrin and dynamin expression after radiation exposure.

Conclusions: Our results suggest that blocking AMONs can enhance the cytotoxicity of CRT. Dynamin mediated endocytosis appears to play a key role in this enhanced cellular update of antisense nucleotides after a sublethal dose of radiation. Since radiation is already part of standard of care cancer therapy, the use of radiation to guide the delivery of systemically administered anti-sense nucleotides is a novel strategy to target key tumor associated proteins for therapeutic benefit key.

Citation Format: Prakash Ambady, Jeffrey Wu, Cymon Kersch, DreeAnna Morris, Michael Pagel, Leslie L. Muldoon, Edward Neuwelt. Radiation enhances the intracellular delivery of anti-MGMT morpholino oligonucleotides (AMONs) to enhance the cytotoxicity of chemo-radiation in cancer cells [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 5087. doi:10.1158/1538-7445.AM2017-5087