Abstract 601: Radiation Therapy Induces Changes in the Endothelial Methylome Affecting Pro-angiogenic Lncrna Expression

Abstract

Radiation therapy is a big part of standard of care in oncology. However, in the long- term patients treated with radiation are at higher risk of suffering cardiovascular events. It is well described that the genotoxic stress induced by Ionizing radiation in normal cardiovascular tissue triggers genetic and epigenetic changes. In addition to coding mRNAs, non-coding RNAs are also highly regulated by changes in methylation and transcription, leading to a tight regulatory response to damage. Genotoxic stress induces global hypomethylation due to decreased expression of DNA methyltransferases (DNMT). We observed that DNMT1, DNMT3A, and DNMT3b are downregulated in response to radiation treatment, in a dose-response manner in human endothelial cells. We have also observed that methylation changes produced by radiation affect a specific ncRNA cluster, DLK1-DIO3. Previous results from our lab indicate that ncRNAs from this cluster are highly responsive to different genotoxic agents, including radiation. Given the essential role of microRNAs and Long non- coding RNAs (lncRNAs) from the DLK1-DIO3 cluster in cardiovascular development and aging, we are interested in understanding how the epigenetic changes induced by radiotherapy affect to lncRNAs expression, and how they influence cardiovascular health in the long term after treatment. We have identified that a specific lncRNA from the DLK1-DIO3 cluster, MEG9, increases in ECs after exposure to ionizing radiation. Interestingly, knockdown of the individual DNMTs enzymes indicates a significant upregulation of MEG9 when DNMT3b is inhibited, more so after radiation treatment. To explore the role of this lncRNA, we performed loss-of-function studies. MEG9 inhibition not only diminished proliferation but also increased apoptosis through caspase 3/7 activation. Consistent with this phenotype, knockdown of MEG9 decreases growth factor-dependent angiogenesis in a 3D fibrin gel angiogenesis assay. Taken together, our findings illustrate how DNA methylation at particular lncRNA loci can regulate their expression and drive endothelial cell fate decisions. Our work illustrates how epigenetic changes may affect the long- term cardiovascular function of cancer patients submitted to radiation therapy.