The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases.

Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more ‘normal-like state’, having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.

Epigenetic modifications such as histone post-transcriptional modifications, DNA methylation, and non-protein-coding RNAs organize the DNA in the nucleus of eukaryotic cells and are critical for the spatio-temporal regulation of gene expression. These epigenetic modifications are reversible and precisely regulated by epigenetic enzymes. In addition to genetic mutations, epigenetic modifications are highly disrupted in cancer relative to normal tissues. Many epigenetic alterations (epi-mutations) are associated with aberrations in the expression and/or activity of epigenetic enzymes. Thus, epigenetic regulators have emerged as prime targets for cancer therapy. Currently, several inhibitors of epigenetic enzymes (epi-drugs) have been approved for use in the clinic to treat cancer patients with hematological malignancies. However, one potential disadvantage of epi-drugs is their lack of locus-selective specificity, which may result in the over-expression of undesirable parts of the genome. The emerging and rapidly growing field of epigenome engineering has opened new grounds for improving epigenetic therapy in view of reducing the genome-wide “off-target” effects of the treatment. In the current review, we will first describe the language of epigenetic modifications and their involvement in cancer. Next, we will overview the current strategies for engineering of artificial DNA-binding domains in order to manipulate and ultimately normalize the aberrant landscape of the cancer epigenome (epigenome engineering). Lastly, the potential clinical applications of these emerging genome-engineering approaches will be discussed.