Targeting oncogenic TERT promoter variants by allele-specific epigenome editing

Background: Activation of dominant oncogenes by small or structural genomic alterations is a common driver mechanism in many cancers. Silencing of such dominantly activated oncogenic alleles, thus, is a promising strategy to treat cancer. Recently, allele-specific epigenome editing (ASEE) has been described as a means to reduce transcription of genes in an allele-specific manner. In cancer, specificity to an oncogenic allele can be reached by either targeting directly a pathogenic single-nucleotide variant or a polymorphic single-nucleotide variant linked to the oncogenic allele. To investigate the potential of ASEE in cancer, we here explored this approach by targeting variants at the TERT promoter region. The TERT promoter region has been described as one of the most frequently mutated non-coding cancer drivers.Results: Sequencing of the TERT promoter in cancer cell lines showed 53% (41/77) to contain at least one heterozygous sequence variant allowing allele distinction. We chose the hepatoblastoma cell line Hep-G2 and the lung cancer cell line A-549 for this proof-of-principle study, as they contained two different kinds of variants, namely the activating mutation C228T in the TERT core promoter and the common SNP rs2853669 in the THOR region, respectively. These variants were targeted in an allele-specific manner using sgRNA-guided dCas9-DNMT3A-3L complexes. In both cell lines, we successfully introduced DNA methylation specifically to the on-target allele of the TERT promoter with limited background methylation on the off-target allele or an off-target locus (VEGFA), respectively. We observed a maximum CpG methylation gain of 39% and 76% on the target allele when targeting the activating mutation and the common SNP, respectively. The epigenome editing translated into reduced TERT RNA expression in Hep-G2.Conclusions: We applied an ASEE-mediated approach to silence TERT allele specifically. Our results show that the concept of dominant oncogene inactivation by allele-specific epigenome editing can be successfully translated into cancer models. This new strategy may have important advantages in comparison with existing therapeutic approaches, e.g., targeting telomerase, especially with regard to reducing adverse side effects.