Development of novel antineoplastic treatment approaches by targeted epigenetic silencing of the vascular endothelial growth factor A and its receptors

Abstract

Decades of efforts of clinicians and scientists to fight against cancer resulted in the development of multiple targeted therapies, which improved clinical outcomes for many types of tumors. Nevertheless, cancer is still the second leading cause of death worldwide, thus more efficient therapeutic approaches are urgently needed. Inhibitors of the VEGFA/VEGFRs axis have shown efficiency against various solid malignancies via reduction of neoangiogenesis of tumors and inhibition of autocrine stimulation of proliferation, migration and invasion of tumor cells by VEGFA. Monoclonal inhibitory antibodies against VEGFA or its receptors efficiently block signaling along the VEGFA/VEGFRs axis, but are very expensive and require repetitive drug injections since they function at the posttranslational level. Targeted epigenome editing is a new emerging technology allowing to control the expression of selected genes at the transcriptional level. Regulation of gene expression is achieved via rewriting of chromatin marks at their cis-regulatory elements. For example, setting of DNA methylation at promoters may lead to stable silencing of corresponding genes. Epigenome editing can be achieved by EpiEditors, artificial chimeric proteins composed of a DNA-binding domain and a DNA methyltransferase, designed to set DNA methylation at target genomic loci. Previous studies demonstrated that methylation of the VEGFA promoter to approximately 50 % resulted in 70 % decrease of VEGFA gene expression in the ovarian cancer cell line SKOV3. This result was promising but only a moderate level of methylation was achieved. Additionally, off-target activity, epigenome editing at non-target genomic loci, has been reported in several studies, and has to be eliminated. This project aimed to develop the technology further to i) improve on-target editing efficiency at the VEGFA promoter to gain higher methylation level; ii) establish multiplex editing to methylate promoters of VEGFA and its receptors VEGFR1, VEGFR2 for simultaneous silencing of all three genes; iii) decrease off-target editing and iv) analyse established DNA methylation patterns.

These goals were approached as follows: i) The DNA genomic targeting technique of EpiEditors was changed from ZPF- to the CRISPR/Cas9 technology. This allowed to employ the recently published EpiEditor composed of the dCas9-10xSunTag protein and the anti-SunTag antibody fused with the highly active DNMT3ACD-DNMT3LCD chimeric methyltransferase. The SunTag allows signal amplification by recruiting up to 10 effector domains, which lead to 40 % higher DNA methylation of the VEGFA promoter compared to the EpiEditors using a single effector domain published previously. ii) Multiplex methylation of the VEGFA, VEGFR1 and VEGFR2 promotors was established for the first time. Targeting of the dCas9-based EpiEditor to multiple loci was realized using vectors expressing several sgRNAs targeting these genes, which theoretically increases editing efficiency compared to co-transfection of vectors expressing single sgRNA used in previous reports. iii) Implementation of dCas9/sgRNA instead of ZFP used previously for targeting the VEGFA promoter significantly improved editing specificity by reducing of off-target effects originating from the DBD. In addition, use of more specific mutant version R887E of EpiEditor showed 5-fold lower off-target activity at a single representative locus. iv) An in-depth analysis of the introduced DNA methylation patterns revealed that the degree of methylation of individual CpG sites depends on several parameters, such as their distance from the sgRNA binding site and the flanking sequence preference of the effector domain. Furthermore, CpG sites can be in hemi- and fully methylated state and the predominance of one or the other state depends on the target locus. The current study led to the development of multiplex epigenome editing of VEGFA and its receptors with the efficiency and specificity superior to previous reports and revealed insights into established DNA patterns which will enhance future design to achieve stable genes silencing and desired antineoplastic therapeutic effects.