Opportunities and Challenges of Epigenetic Editing in Human Diseases
Goubert, Désirée
DOI:
10.33612/diss.173201281
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Publication date: 2021
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Goubert, D. (2021). Opportunities and Challenges of Epigenetic Editing in Human Diseases: Towards the Curable Genome. University of Groningen. https://doi.org/10.33612/diss.173201281
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General Introduction
An introduction to Epigenetics
Epigenetics is a layer on top of the DNA that causes heritable, yet reversible changes in gene expression, without affecting the primary DNA sequence (1, 2). Epigenetic gene regulation is very stable but also highly flexible. Epigenetic states can persist over many cell generations, whilst on the other hand epigenetic regulations make sure that genes can dynamically respond to external signals (3). During mammalian development cellular differentiation is regulated by the epigenetics. Even though most cells within complex multicellular organisms contain the same DNA, there are many different cell types, making up the various tissues and organs. Epigenetic modifications underlie cell identity by switching cell-type specific genes on or off (4, 5). Furthermore, this regulation of gene expression patterns is maintained throughout cell divisions (e.g. proliferating liver cells, remain liver cells) and can even be inherited by the offspring (6, 7).
Epigenetic modifications are chemical or physical changes of the DNA and chromatin catalysed by enzymes in a highly dynamic way. Epigenetics represents a molecular explanation as to why healthy living and making responsible choices is so important and has even been defined as “The missing link between nature and nurture” (8, 9). Epigenetic marks instruct gene expression patterns by stimulating or denying transcription of genes through regulating access of transcription factors (TFs), transcriptional initiation complexes, and RNA polymerase to DNA (10). The accessibility of DNA thus causes densely packed genes not to be expressed (or less efficiently), whilst a loosely packed gene will lead to an increased expression. The epigenetic signatures, which are unique for each cell-type, are placed by epigenetic enzymes that write or erase epigenetic marks. These marks include methylation of cytosine (described in detail in Chapter 3) and a wide variety of posttranslational
modifications of histone proteins, where the DNA is wrapped around. Epigenetic marks cause functionally relevant changes to the genome that do not involve changes in the primary DNA sequence (11, 12).
Epigenetics in human diseases
Disruptions in the epigenetic layer can have severe consequences on gene expression and cellular behaviour and contribute to the development of many different diseases (13-16) as well as sensitivity to treatment and development of resistance (17, 18). In cancer for example, tumour suppressor genes, which normally block cancer growth, are often DNA hypermethylated, which is associated with a silenced gene expression state (19). Unravelling the link between DNA methylation and cancer progression is aiding to the development of clinical applications, for example by using site specific presence or absence of DNA methylation as a diagnostic marker (20, 21). As epigenetic changes are a naturally occurring and reversable process, they present an attractive target for therapeutic intervention. It is therefore not surprising that from 2006 onwards so called epi-drugs have entered the clinical area (22). These drugs target epigenetic enzymes that write or erase epigenetic marks. Such epi-drugs include DNA methyltransferase inhibitors (DNMTIs: e.g. 5-azacytidine and decitabine), histone deacetylase inhibitors (HDACIs: e.g., vorinostat, Zolinza and romidepsin) and histone methyltransferase inhibitors (HMTIs) and they are used in the clinic for the treatment of haematological malignancies, solid tumours and neurological disorders (22-33). Unfortunately, wide-spread clinical application of epi-drugs is hampered by the fact that these drugs act genome-wide rather than in a gene-specific manner, causing unwanted effects, including upregulation of pro-metastatic genes (34, 35) or of genes encoding drug resistance-associated proteins (36).
Epigenetic Editing
Therefore, more targeted epigenetic approaches are desirable and one such approach is Epigenetic Editing. In this approach, which is described in more detail in Chapter 2, epigenetic
marks are placed or removed from specific target genes, without changing the primary DNA sequence (37-43). Artificial Transcription Factors (ATFs), combined with DNA binding platforms, can also be used to modulate gene expression in a targeted way (44-47). Such ATFs represent transient transcriptional repressors (e.g. super KRAB domain (SKD)) (48, 49) or transient transcriptional activators (e.g. the tetramer of herpes simplex virus protein VP16 (VP64)) fused to DNA binding domains. In contrast to ATFs, epigenetic writers or erasers
change the epigenetic modifications at a target gene, and are therefore more likely to be maintained by cellular processes even after removal of the Epigenetic Editing tool (50-52). In comparison to epigenetic enzymes, ATFs do not possess any enzymatic activity on their own, but they recruit other epigenetic players to have an effect on gene expression. Epigenetic Editing tools consist out of at least two components: a DNA binding component, which functions as a molecular GPS, guiding the tool towards the intended genomic location; and an effector domain (ED), which places or erases epigenetic marks. The two DNA-binding platforms used in this thesis are the CRISPR-dCas9 platform (53) and Zinc Finger (ZF) proteins. CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats and has been first reported as a bacterial immune response against viruses. Every time a bacterium gets invaded by a virus short DNA copies are made of the viral DNA. The RNA products of these DNA copies, called single guide RNAs (sgRNAs), recognise the viral DNA when it invades a second time and recruit the CRISPR binding partner Cas9. Cas9 then makes a double stranded cut, disabling the DNA and thereby inactivating the virus, rendering it harmless. Since 2012 Jennifer Doudna and Emmanuelle Charpentier demonstrated that it is possible to use this mechanism in human cells for bioengineering by delivering the Cas9 enzyme together with a sgRNA that is designed to target a gene of interest (54). Currently, CRIPSR-Cas9 is used in clinical trials for gene editing purposes, mostly ex vivo (patient cells are taken out of the body, treated with gene editing compounds, and put back into the patient) (55-58), but recently also for the first time in vivo (in this case administrated to the eye of a patient) (59). In gene editing, the Cas9 enzyme is targeted towards a mutated gene, where it makes a cut, rendering the gene inactive and incapable of performing its harmful function (Figure 1). It is also possible to insert a correct gene by providing the cell, after Cas9 DNA cleavage, with a correct piece of DNA. This corrected gene is inserted in the cleaved DNA via its homologues overhangs with the cleaved site and used to repair the cut (60).
The CRISPR-dCas9 platform used for Epigenetic Editing consists of the same DNA-binding component, in the form of sgRNAs, however the Cas9 enzyme has been deactivated. Two mutations introduced into the Cas9 enzyme render it incapable of making a cut, but it still can be used as a molecular shuttle bringing for example an epigenetic ED to a specific location within the genome. Epigenetic EDs can then affect the epigenetic environment of that specific
gene, and increase or decrease its expression. ZF proteins are small naturally occurring transcription factors, forming the largest group of all transcription factors in the human genome (61), that can also be fused to epigenetic EDs or ATFs and designed to target gene-specific DNA sequences (44, 62, 63). The relatively small size and low immunogenicity of ZF proteins are an advantage compared to other DNA-targeting proteins (39, 63).
Figure 1: Schematic representation of gene editing using CRISPR-Cas9. The Cas9 enzyme inactivates the
mutated target gene by making a cut in the DNA (Left side) or corrects a gene when provided with the correct donor DNA (Right side) sgRNA: Single Guide RNA.
Targeted overwriting of epigenetic marks using these platforms holds promise to permanently reprogram gene expression and has successfully been applied in diverse therapeutic models (64, 65). For example, Thakore et al. successfully silenced the transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of inactivated viral particles carrying dCas9-KRAB and sgRNAs targeting of the
Pcsk9 gene led to a moderate immune response and an efficient repression of Pcsk9 (64). Xu et al. showed that in vivo reactivation of several anti-fibrotic genes is also possible using
Epigenetic Editing. Targeted demethylation using TET enzymes led to specific gene reactivation and attenuation of fibrosis (65). Epigenetic Editing thus has the potential to develop into clinically relevant one-and-done approaches, where patients are treated once, after which the modified epigenetic marks are remembered and maintained on the target
gene, and could lead to breakthroughs in curing diseases that are currently uncurable: “The curable epigenome”.
Epigenetics in the development of endocrine therapy resistance
One of the diseases that this thesis focuses on is breast cancer, more specifically on oestrogen receptor alpha (ERα) positive (ER+) breast cancer (which accounts for 70% of all breast cancer cases). These ER+ tumours depend on the female growth hormone oestrogen for their growth and can be treated with endocrine therapy (66). Endocrine therapy in a premenopausal setting makes use of drugs that selectively modulate oestrogen receptor binding (e.g. Tamoxifen) or downregulate the oestrogen receptor (e.g. Fulvestrant), in a postmenopausal setting drugs are used that inhibit the synthesis of the hormone oestrogen (e.g. Aromatase inhibitors such as Letrazol). Unfortunately, over 40% of patients who receive endocrine therapy eventually develop resistance to the treatment (67), which is accompanied by poor prognosis (17). For therapy resistance to arise, a tumour cell undergoes molecular changes, or has intrinsically present characteristics that allow its continuous cell proliferation under treatment conditions, providing these resistant tumour cells with a selective advantage. Acquired resistance to endocrine therapy is a long-term process in which genetic alterations are expected to act synergistically with epigenetic changes (17). Epigenetic reprogramming has been shown to contribute to the development of endocrine therapy resistance by inducing changes in gene regulatory networks (68-70). For example as shown by Magnani et
al. endocrine therapy resistant cells activate lipid synthesis (endogenous cholesterol) through
alterations in epigenetic histone modifications, both in resistant breast cancer cells and in patients (69). The increased cholesterol synthesis caused an activation of the oestrogen receptor, circumventing the reliance of the cells in the presence of oestrogen.
Also other epigenetic mechanisms including DNA hypermethylation, overexpression of histone deacetylases, enhancer regulation, non‑coding RNAs, chromatin remodelling, post‑translational histone modifications and histone variants have been shown to contribute to the development of endocrine therapy resistance (71). Stone et al. showed that DNA hypermethylation in endocrine resistant cells mainly occurred at oestrogen-responsive enhancers (72). Enhancers are regulatory DNA sequences that can alter the transcription of
an associated gene when they are activated. DNA methylation of these enhancers led to reduced oestrogen receptor binding and subsequently reduced expression of key regulators of ERα activity. Furthermore, significantly higher DNA methylation levels in these enhancers were also noted in breast cancer patients that relapsed from endocrine treatment (72). Yamamoto et al. investigated the role of the histone H3 lysine 4 (H3K4) demethylase JARID1B in luminal breast tumours (73). They showed that JARID1B is frequently amplified and overexpressed in luminal breast cancer and that a high JARID1B activity is associated with resistance to endocrine therapy and a worse clinical outcome. An additional level of epigenetic complexity is added by histone variants, which can replace original histones and contribute to changes in compaction of the chromatin structure. Both histone variants H2A.Z (an H2A variant) and HIST1H2BE (an H2B variant) have been linked to endocrine resistance (74, 75). H2A.Z overexpression led to an increased proliferation under oestrogen-deprived conditions (74) and HIST1H2BE was overexpressed in endocrine-resistant cell lines, as well as in tumours from patients that relapsed after aromatase inhibitor treatment (75).
These and other studies show the high urge to further identify which epigenetic changes are responsible for the development of endocrine therapy resistance and to develop diagnostic (71) and therapeutic tools (27). To this end, the international research consortium called EpiPredict “Epigenetic regulation of endocrine therapy resistance in breast cancer”, was initiated of which this PhD project is part. This innovative training network (ITN) is funded by EU H2020 MSCA-ITN-2014 and coordinated by Dr PJ Verschure, UvA, NL. In this consortium, 12 PhD students from eight different countries and scientific institutes have studied the role of epigenetic regulation in resistance development for endocrine therapy in ER+ breast cancer. My specific role as ”Early Stage Researcher number 8” (ESR8) from Work Package 5 was to design, construct and test gene-specific epigenetic interference tools to prevent and/or reverse endocrine therapy resistance. Together with the scopes of EpiPredict this is further elaborated on in Chapter 2.
As mentioned earlier, one of the epigenetic marks, and perhaps also the most well-known one, is DNA methylation, and changes in DNA methylation have been reported in multiple tumours, including breast cancer (76, 77). DNA methyltransferases are the enzymes responsible for adding methyl groups to cytosine bases and promoter regions of genes where
a lot of cytosines (CpG island, CGI) are methylated are associated with transcription inhibition (78). Chapter 3 explores DNA methylation and writing of this specific epigenetic signature.
Available tools for editing DNA methylation are described as well as their applications. In addition, it is explained how to achieve targeted DNA methylation/demethylation and the advantages and disadvantages of this approach are discussed.
Using Epigenetic Editing to achieve functional effects
The ultimate goal of Epigenetic Editing is to achieve changes in cell functioning, for example to render cells less resistant to a therapy. In Chapter 4 the ability of Epigenetic Editing to
accomplish functional changes was explored in different breast cancer cell subtypes, in which the oncogenic potential of the PLAU gene was investigated. Experimental inhibition of the
PLAU gene has previously been shown to reduce tumour growth, aggressiveness and
metastasis. As these experimental methods are not yet clinically possible, other compounds have been investigated to inhibit PLAU. One such promising compound is plant-derived Withaferin A (WA), a natural compound with wide-ranging pharmacological activities including cardio-protective, inflammatory, immuno-modulatory, angiogenic, anti-metastatic and anti-carcinogenic effects (79, 80). WA decreases the expression of PLAU, but also of other genes involved in cell adhesion, inflammation, and metastasis in vitro, as well as
in vivo (81-84). Furthermore, several studies have shown that DNA methylation is essential to
control PLAU expression (85-88). We therefore set out to use Epigenetic Editing as a method to manipulate PLAU expression and investigate the functional impact of PLAU in a targeted approach.
Sustainability of Epigenetic Editing
One of the early-time dogmas about Epigenetic Editing is its ability to lead to long-term, sustained effects, and there is still contradiction in the field whether this is possible. Some reports show sustained epigenetic reprogramming through Epigenetic Editing (52, 89-92), whilst this could not be repeated for other genes in different contexts (93, 94). The current consensus states that for sustained epigenetic reprogramming multiple EDs are required for long-term repressive effects (95-98). For clinical applications however, the requirement of
multiple components would be a serious limitation. In Chapter 5 we tested whether the
fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor targeted to the PLOD2 promoter via ZF- or CRISPR-dCas9-mediated targeting. KRAB is an ATF, which are generally considered to act transient in somatic cells (43, 44, 52, 95-97, 99), whilst for the induction of stable heterochromatin, direct editing of epigenetic marks, e.g. DNA methylation, is assumed to be more effective (90, 100). Here, we tested these two EDs for their capability to induce direct and indirect epigenetic modifications, (long-term) gene repression and their effect on transcription when having induced a heterochromatic state.
Technical improvements
Like with all novel techniques, there are quite some challenges to overcome when optimizing and validating Epigenetic Editing. Therefore, a big part of this PhD project focussed on improving transfection efficiency, delivery of the constructs to the cells and the ease of read-outs of laboratory systems. In Chapter 6, different approaches (e.g. the MS2 system,
fluorescent assisted cell sorting and several methyltransferase variants) are applied to modulate the expression of UCHL1, a gene with relevance in cancer and airway diseases. By overcoming technical hurdles, our understanding of underlying biological phenomena can increase even further, and in this way Epigenetic Editing can rise to its full potential as a flexible one-and-done research tool with various clinical applications.
One way assumed to overcome the problem of delivery in a laboratory setting is to make stable cell lines, a method that has been optimized and described in detail in chapter 7.
Briefly, in this procedure cells are infected with lentiviruses encoding dCas9 fused to an ED. This construct will randomly integrate into the cell’s genome, and will be expressed continuously. The gene-specific sgRNAs can then be transfected transiently to assess long-term effects of modified epigenetic marks on gene expression and cellular behaviour. As the plasmids for sgRNAs are much smaller than the dCas9-ED containing plasmids, transfection efficiency is expected to increase compared to transiently transfecting both the sgRNAs and the dCas9-ED. Furthermore, it might be essential to transfect sgRNAs targeted towards
multiple genes, which is of importance in diseases like e.g. cancer where multiple genes are involved.
Ways ahead
Even though there are still a lot of challenges to overcome by future researchers within this field, every piece of information is valuable and gets us a bit closer to using Epigenetic Editing in a clinical setting and a step closer towards a curable genome. There are rapid developments in epi-drug discovery (22), mainly focused on targeting the epigenetic enzymes (readers, writers and erasers of epigenetic marks) using small-molecule inhibitors (101-103) and a wide range of epigenetic-based drugs are undergoing clinical trials. Epigenetic drugs that are used in the clinic however, are mostly limited to haematological malignancies (104, 105). Many other diseases could benefit from the potential of epigenetic drugs (106-109), but they would require more targeted approaches. In this respect, Epigenetic Editing provides the possibility to target many different epigenetic enzymes to a specific locus of interest to rewrite epigenetic marks in a sustained manner. Furthermore, ongoing trials using DNA-targeting approaches (Sangamo trials, explained in more detail in the general discussion of this thesis), are giving us valuable information with regard to delivery, efficiency and safety of gene targeting. This knowledge can thus be used to translate Epigenetic Editing into a clinical applicable tool. The future is therefore looking bright in the field of targeted epigenetic approaches.
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