Drug Resistance Updates 32 (2017) 16–22

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Drug Resistance Updates

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In cancer, A-to-I RNA editing can be the driver, the passenger, or the MARK
mechanic
⁎
Nabeel S. Ganem1, Noa Ben-Asher1, Ayelet T. Lamm
Faculty of Biology, Technion - Israel Institute of Technology, Technion City, Haifa 32000, Israel

A R T I C L E I N F O A B S T R A C T

Keywords: In recent years, A-to-I RNA modifications performed by the Adenosine Deaminase Acting on RNA (ADAR)
ADAR protein family were found to be expressed at altered levels in multiple human malignancies. A-to-I RNA editing
Chemotherapeutic drug sensitivity changes adenosine to inosine on double stranded RNA, thereby changing transcript sequence and structure.
Cancer biomarkers Although A-to-I RNA editing have the potential to change essential mRNA transcripts, affecting their corre-
Gene therapy
sponding protein structures, most of the human editing sites identified to date reside in non-coding repetitive
transcripts such as Alu elements. Therefore, the impact of the hypo- or hyper-editing found in specific cancers
remains unknown. Moreover, it is yet unclear whether or not changes in RNA editing and ADAR expression
levels facilitate or even drive cancer progression or are just a byproduct of other affected pathways. In both
cases, however, the levels of RNA editing and ADAR enzymes can possibly be used as specific biomarkers, as
their levels change differently in specific malignancies. More significantly, recent studies suggest that ADAR
enzymes can be used to reverse the oncogenic process, suggesting a potential for gene therapies. This review
focuses on new findings that suggest that RNA editing by ADARs can affect cancer progression and even for-
mation. We also discuss new possibilities of using ADAR enzymes and RNA editing as cancer biomarkers, in-
dicators of chemotherapeutic drug sensitivity, and even to be themselves potential therapeutic tools.

Introduction to A-to-I RNA editing
A-to-I RNA editing was discovered 30 years ago and is one of the
most studied transcriptome modifications (Bass and Weintraub, 1987;
Rebagliati and Melton, 1987). However, only in the last few years has
A-to-I RNA editing been possibly implicated in cancer formation and
progression. Specifically, A-to-I RNA editing is the deamination of
adenosine to inosine on double stranded RNAs (dsRNAs) (Fig. 1A),
which is performed by ADAR (Adenosine Deaminase Acting on RNA)
protein family (Bass, 2002). Inosine residues are recognized by the
cellular machinery as guanosine. Therefore, A-to-I editing in coding
regions can recode protein sequences and generate proteins with new
functions (reviewed in (Nishikura, 2016)). Currently, there are three
known ADAR proteins in mammals, ADAR1, ADAR2 and ADAR3.
ADAR1 and ADAR2 are ubiquitously expressed, whereas ADAR3 is
exclusively expressed in the brain (Melcher et al., 1996a; Melcher et al.,
1996b). All ADAR proteins have two main domains, a catalytic dea-
minase domain at their C-terminal region and a number of dsRNA
binding domains (dsRBD) at their N-terminal region ((Bass, 2002),
(Thomas and Beal, 2017), Fig. 1B). The ADAR3 catalytic deaminase
domain is probably non-functional (Melcher et al., 1996a). ADAR1 also
has Z-DNA binding domains (ZDBD), whose functional activity is cur-
rently unknown (Herbert et al., 1997).
In addition, there are two more ADAR-related genes in mammals,
ADAD1 and ADAD2, which are exclusively expressed in the testis and
are enzymatically inactive with a yet unknown function (Schumacher
et al., 1995). The ADAR1 gene encodes two main isoforms from alter-
native promoters, ADAR1p110 and ADAR1p150 (Fig. 1B). ADAR1p110
is the shorter isoform. It is constitutively expressed and mainly loca-
lized in the nucleus. In contrast, ADAR1p150 is mainly cytoplasmic and
its expression is induced by the interferon pathway (Patterson and
Samuel, 1995; Liddicoat et al., 2016). The ADAR2 gene is mostly ex-
pressed in the brain and is localized in the nucleus (Maas and
Gommans, 2009). ADAR1 and ADAR2 were shown in vitro to homo-
dimerize (Cho et al., 2003) and they differ in their editing targets.
Specific editing sites that alter proteins were mainly found in the brain.
One of the most studied edited transcript is that of glutamate ionotropic
receptor AMPA type subunit 2 (GRIA2) (Sommer et al., 1991), which is
part of a family of glutamate receptors that are sensitive to α-amino-3-
hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and function as

⁎
Corresponding author.
E-mail address: ayeletla@technion.ac.il (A.T. Lamm).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.drup.2017.09.001

1368-7646/ © 2017 Elsevier Ltd. All rights reserved.

N.S. Ganem et al.
ligand-activated cation channels. These channels are assembled from 4
related subunits, GRIA1-4. RNA editing in GRIA2 is thought to render
the channel impermeable to Ca+2. Codon editing in GRIA2 subunit,
which in some cases is edited to an extent of nearly 100% in transcripts,
is needed to keep neuronal Ca2+ homeostasis. Indeed, mice harboring
mutations in ADARs that lack the ability to edit these transcripts exhibit
an early onset of epileptic seizures and premature death (Brusa et al.,
1995). RNA editing can also alter splice sites and eliminate stop codons.
Interestingly, ADAR2 edits its own pre-mRNA, resulting in a novel
splice site that leads to a truncated protein due to the formation of a
premature stop codon (Feng et al., 2006).
In recent years, advances in high-throughput sequencing led to the
discovery that most editing events occur in clusters in non-coding re-
petitive transcripts, termed ‘hyperediting’, primarily in Alu repeat ele-
ments. In fact, editing events occur in more than half of the human
transcriptome (Bazak et al., 2014). The level of editing (i.e., percentage
of transcripts undergoing editing in a particular site) at site-specific
coding sites is higher than that usually found in ‘hyperediting’ sites and
is mostly associated with ADAR2 function.
Generation of microRNA can also be affected by A-to-I editing. Some
pri-microRNAs undergo RNA editing and this editing prevents their
processing and affects their ability to produce mature microRNAs
(miRNAs) (Kawahara et al., 2007a; Yang et al., 2006). miRNAs can be
edited at their seed regions, which can affect their binding efficiency to
their targets and even alter their target specificity (Kawahara et al.,
2007b). Editing in the target miRNA binding site can also affect the
efficiency of the miRNA binding and regulate the target expression (Cho
et al., 2017).
A-to-I RNA editing is essential in mice and humans. Mutations in
ADAR genes as well as mutations that affect ADAR’s editing function-
ality are associated with several human diseases. Mutations in the
ADAR1 gene, mainly in its deaminase domain, are associated with the
pathogenesis of dyschromatosis symmetrica hereditaria (DSH; OMIM #
127400). The latter is a rare autosomal dominant skin disorder char-
acterized by hyperpigmented and hypopigmented macules on the face
and dorsal aspects of the extremities that appear in infancy or early
17
Drug Resistance Updates 32 (2017) 16–22
Fig. 1. Schematic representation of the deamination
reaction and human ADAR protein family. (A) An
amine group removal leads to the conversion of
adenosine to inosine by an ADAR enzyme. Guanosine
is shown to illustrate the structural similarity with
inosine. (B) Members of the human ADAR family.
The two main isoforms of ADAR1 are presented,
ADAR1 p110 and ADAR1 p150. The deaminase do-
mains are colored in green, the RNA binding do-
mains (RBD) are colored in red and Z DNA binding
domains (Z −DBD) are colored in blue. Proteins are
scaled. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web
version of this article.)
childhood (Zhang et al., 2004). ADAR1 mutations are also associated
with Aicardi-Goutières syndrome (AGS; OMIM # 225750), an auto-
somal dominant autoimmune disorder characterized in its most severe
form by cerebral atrophy, leukodystrophy, intracranial calcifications
and chronic cerebrospinal fluid (CSF) lymphocytosis. This syndrome
results from the chronic activation of type I interferon (Rice et al.,
2012). Since editing that affects protein structure is mostly abundant in
the nervous system, it is not surprising that changes in transcript editing
levels were shown to result in neurological disorders including: Alz-
heimer’s disease (Gaisler-Salomon et al., 2014), amyotrophic lateral
sclerosis (ALS) (Hideyama et al., 2010), and autism spectrum diseases
(Eran et al., 2013).
In the current review, we focus on the involvement of ADARs and A-
to-I editing in cancer, including the ability to use editing sites as di-
agnostic biomarkers, and new developments of using ADAR proteins as
druggable targets for cancer therapeutics.
The function of ADARs in development and immunity
Given the substantial amount of editing found in the human tran-
scriptome, it is not surprising that RNA editing by ADARs has essential
roles. Both ADAR1 and ADAR2 are required for normal development
and knockout mice for either of these genes die early in development
(Higuchi et al., 2000; Wang et al., 2000). The early embryonic lethality
in ADAR2 knockout mice is partially due to the lack of editing in GRIA2
RNA, a glutamate ionotropic receptor (see above, (Higuchi et al.,
2000)). ADAR1 knockout mice phenotypes led to our understanding of
its possible important role in the innate immune response (Hartner
et al., 2004; Wang et al., 2004). ADAR1 was found to be a key player in
the suppression of the antiviral dsRNA sensing type I interferon
pathway (Liddicoat et al., 2015) by preventing self-RNAs from trig-
gering the innate immune response (George et al., 2016). ADARs can
also be pro-viral for specific viruses (reviewed in (Samuel, 2012)).
ADAR1 is also induced by the interferon response (Patterson and
Samuel, 1995), which might be linked to the chronic inflammatory
state observed in several types of cancer (Fumagalli et al., 2015). ADAR
N.S. Ganem et al.
and A-to-I RNA editing functions in organismal development and its
immunity were shown in invertebrates and other organisms as well
(reviewed in (Ganem and Lamm, 2017)).
ADAR enzymes and A-to-I editing in cancer
For many years, cancer research focused on mutations in genes that
can lead to cancer initiation and progression, including mutations that
cause cells to proliferate without constraints, mutations that affect cell
cycle checkpoints, and mutations that induce further mutations by
impairing mechanisms of DNA damage repair. Epigenetics mechanisms
were also shown to be important in formation of neoplasia and their
abnormalities, including alterations in methylation patterns or chro-
matin remodeling, which are common in many cancer types (Sharma
et al., 2010). Therefore, it was predicted that A-to-I editing would be
significantly altered in cancer.
The expression of ADARs and the corresponding editing capacity
have been studied in various cancer types, revealing distinct patterns in
different tumors. In glioblastoma multiforme (GBM), the most ag-
gressive type of central nervous system tumor, lower ADAR2-mediated
editing was observed, specifically in GRIA2 mRNA, without a corre-
sponding downregulation of ADAR2 expression (Maas et al., 2001). In
vitro and in vivo experiments in which ADAR2 was overexpressed re-
versed the malignant phenotype of glioblastoma cells (Cenci et al.,
2008; Galeano et al., 2013). In contrast, both ADAR1 and ADAR3 were
found to be upregulated in brain tumors (Cenci et al., 2008; Oakes
et al., 2017), while a different study found that all ADAR gene ex-
pression levels were reduced (Paz et al., 2007). Interestingly, between
the two ADAR1 isoforms, only the p110 isoform is expressed in GBM,
since a deletion caused by a splicing variant in the p150 isoform creates
a frameshift mutation (Cenci et al., 2008). The lower editing rate in
glioblastoma might result from inhibition of ADAR2 editing by the
overexpressed ADAR1, which was shown to inhibit ADAR2 in vitro, in a
dose-dependent manner (Cenci et al., 2008). ADAR3 overexpression
might also be involved as it was shown to inhibit GRIA2 editing, pos-
sibly by competing with ADAR2 on GRIA2 mRNA binding (Oakes et al.,
2017). Another possibility is that a splicing variant of ADAR2 is ex-
pressed, which harbors a diminished catalytic activity (Maas et al.,
2001). ADAR2 was also shown to alter the expression and editing levels
of microRNAs that are involved in tumorigenesis in glioblastoma cells
(Tomaselli et al., 2015).
Apart from solid tumors, in chronic myeloid leukemia (CML), higher
expression of the inflammatory INF-related pathway and ADAR1p150
isoform were observed (Jiang et al., 2013). Expression of ADAR1p110,
which is not dependent on the inflammation signaling axis, was un-
changed. In more advanced leukemias, more A-to-I RNA editing was
observed, mainly in Alu-repeat sequences (Paz-Yaacov et al., 2015). In
vitro studies showed that overexpression of ADAR1p150 drives hema-
topoietic differentiation towards the myeloid lineage, while ADAR1
knockout impairs malignant myeloid progenitor self-renewal (Jiang
et al., 2013; Steinman et al., 2013).
In hepatocellular carcinoma (HCC), a disrupted editing balance has
been observed in tumor tissues by RNA-seq analysis (Chan et al., 2014).
In addition, ADAR1 upregulation and ADAR2 downregulation corre-
lated with increased risks of liver cirrhosis and occurrence of hepato-
cellular cancer (Chan et al., 2014).
ADAR1 was shown to edit antizyme inhibitor-1 (AZIN1) mRNA and
cause amino acid composition change in the protein (Chen et al., 2013).
Antizyme inhibitors are homologs of ornithine decarboxylase (ODC, the
key enzyme in polyamine biosynthesis), which are devoid of ornithine
decarboxylation activity. Hence, antizyme inhibitors function as posi-
tive regulators of polyamine levels by sequestering antizymes (the latter
block the key enzyme in polyamine biosynthesis, ODC) and thereby
neutralize their effect. AZIN1 editing causes an increase in cell pro-
liferation and high levels of edited AZIN1 were suggested to be a driver
of HCC (Chen et al., 2013).
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Drug Resistance Updates 32 (2017) 16–22
In metastatic malignant melanomas, ADAR1 is downregulated while
ADAR2 levels are unchanged (Nemlich et al., 2013). This change in
expression is accompanied by altered expression of many other genes
and miRNAs, most of which do not undergo RNA editing. In vitro,
ADAR1 silencing enhances the malignant phenotype, while conversely,
overexpression of ADAR1 inhibits melanoma cells proliferation
(Nemlich et al., 2013). This inhibition is independent of ADAR1 editing
activity as catalytically-inactive proteins displayed the same effect as
the active protein. Downregulation of ADAR1 in melanoma has been
shown to be mediated by overexpression of two miRNAs that target
ADAR1, miR-17 and miR-432 (Nemlich et al., 2013). High level ex-
pression patterns of ADAR1 with no changes in the expression of
ADAR2 are found in esophageal squamous cell carcinoma (ESCC) (Qin
et al., 2014). Higher expression levels of ADAR1 and higher actual
editing levels have been correlated with poor ESCC patient prognosis
(Qin et al., 2014).
While the studies in the abovementioned cancers concentrated on
specific tumor and editing targets, three recent studies comprehensively
analyzed the role of ADARs in cancer by using the data of Cancer
Genome Atlas (TCGA) collection (Fumagalli et al., 2015; Han et al.,
2015; Paz-Yaacov et al., 2015). They found that in most cancer types,
editing levels are higher than in the normal paired tissues (see examples
in Fig. 2). While all ADAR family members displayed changes in ex-
pression in many tumors, ADAR1 seems to be the most important family
member in the context of cancer, as alterations in its expression were
observed in all the tumors tested. Differential expression is achieved by
distinct modalities: gene amplification, alternative splicing and down-
regulation through miRNAs. The opposite effects that ADAR1 has on
different tumor types can be attributed to different targets of RNA
editing and different downstream effectors in the different tissues.
One open question that comes up in many cancer studies, is whether
or not these differences between cancer cells and their paired normal
cells are a byproduct of the many genomic abnormalities that occur in
cancer progression (passenger alterations) or whether the observed
differences are the drivers of cancer progression. Concerning RNA
editing, the answer is yet unclear. Changes in gene editing levels that
are linked to cancer and in cancer cell microRNA binding sites (Han
et al., 2015; Paz-Yaacov et al., 2015), might be driver alterations
events. Another indication that ADAR1 overexpression might drive
cancer is that upon knockdown of ADAR1 expression in breast cancer
cells, a decrease in cell proliferation and an increase in apoptosis is
observed (Fumagalli et al., 2015). However, ADAR1 expression is
known to be induced by the type 1 interferon response (Liddicoat et al.,
2016; Rice et al., 2012), which is related to the chronic inflammatory
state commonly seen in cancer. Therefore, the changes in RNA editing
might be a result of the inflammatory environment of the tumors, which
constitutes a passenger position (Fumagalli et al., 2015).
In addition to showing that cancer cells differ in their ADAR activity
from normal cells, these studies have also raised the possibility of using
RNA editing levels as cancer biomarkers and ADAR enzymes as bona
fide drug targets. These possible therapeutic avenues are discussed
below.
A-to-I editing as cancer markers
Cancer diagnosis, staging of the disease and the patient’s response to
therapy can be predicted by using unique and reliable biomarkers.
These biomarkers can be specific to a certain type of cancer or can be
present in several cancer types. Some tumor biomarkers are proteins
that are present on normal and cancer cells or are secreted into the
body’s fluids (Mehta et al., 2010; Petricoin et al., 2006; Sawyers, 2008).
mRNA levels of specific transcripts, epigenetics changes, and genetic
variations are also common biomarkers. However, in some cases, the
levels of specific tumor markers can be elevated under benign condi-
tions from unknown reasons. In addition, a specific cancer type can
express distinct biomarkers in different patients due to cancer
N.S. Ganem et al. Drug Resistance Updates 32 (2017) 16–22

Fig. 2. Altered ADAR expression and RNA editing levels in human cancers. The scheme presents some findings of changes in ADAR expression (mainly ADAR1) and RNA editing levels in
specific tumors. Alterations in ADAR expression and RNA editing levels are presented by blue and red arrows, respectively. Arrows pointing upwards denote upregulation and arrows
pointing downwards represent downregulation. Data was taken from the following studies: (1) (Han et al., 2015), (2) (Paz-Yaacov et al., 2015), (3) (Fumagalli et al., 2015), (4) (Qin et al.,
2014). Cancer types that appear in black are common to both genders. Prostate adenocarcinoma (PRAD), a male-gendered cancer, appears in blue. Breast cancer, which is predominantly
found in females, appears in pink. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

heterogeneity originating from genomic instability (Mehta et al., 2010;
Sawyers, 2008). Clearly however, cancer biomarkers are not available
yet for various cancer types. This stresses the importance of identifying
reliable markers for classifying cancer types and for evaluating the
patient’s response to certain drug treatments (Mehta et al., 2010;
Sawyers, 2008). In this respect, Paz-Yaacov et al. (Paz-Yaacov et al.,
2015) have examined the possibility of using editing levels as a novel
cancer biomarker. They determined editing levels in Alu sequences in
several cancer types and found a correlation between low editing levels
in Alu and higher survival rates in head and neck, liver, and breast
cancers. Hyper-editing in Alu might reflect loss of genomic stability and
therefore a more advanced tumor progression (Paz-Yaacov et al., 2015).
However, this finding is currently restricted to certain types of cancer as
some cancer types show hypo-editing (Han et al., 2015). Moreover, as
shown above, altered editing levels in specific transcripts can also re-
port on the cancer state. Wang and colleagues did a similar screen using
the TCGA dataset to study the correlation of editing levels in cancer
types using editing in miRNAs as markers (Wang et al., 2017). They
found that editing “hot-spots” in miRNAs are associated with estab-
lished tumor subtypes, cancer stage, and patient survival time. Editing
in miRNAs can affect their target selection, which can be for instance
oncogenes and tumor suppressor genes. For example, editing in miR-
200b, a key tumor metastasis suppressor, correlates with patient
prognosis and might serve as a strong biomarker (Wang et al., 2017).
RNA editing and chemotherapeutic drug sensitivity
While A-to-I RNA editing may be possibly used for cancer diag-
nostics and prognosis, it was recently suggested that when editing
events are dysregulated in cancer, they could target and affect many
pathways; hence, the latter may serve as possible druggable targets for
development of novel therapeutic interventions (Han et al., 2015). This
opens the possibility to predict the impact of cancer treatments in in-
dividual patients based on specific transcript editing patterns. To ex-
plore the RNA editing impact on drug sensitivity, Han et al. (Han et al.,
2015) performed cell viability assays on tumor cells after introducing
mutations in genes that mimic the tumor’s unique RNA editing events
such as AZIN1S367G, GRIA2R764G, and COG3I635V. They found an
increase in cell survival, which suggests that these editing events have a
direct effect on cancer progression. Next, to explore the possibility that
these editing events can affect the cancer cells’ response to che-
motherapeutic drugs, they did a cytotoxicity screen and found that
editing events can affect cancer cell sensitivity to several compounds
used for targeted therapeutics. Using the Cancer Cell Line Encyclopedia
(CCLE) (Barretina et al., 2012), they also found a correlation between
nonsynonymous RNA editing levels and anticancer drug sensitivity.
It is also important to note that drugs can also affect the expression
levels of ADAR enzymes thereby bringing about hyper- or hypo-editing
events and possibly editing events in positions that were not edited
before. For example, cocaine can lead to reduced ADAR2 expression
and subsequently diminished editing activity (Schmidt et al., 2015).
Antidepressant drugs were found to cause alteration in RNA editing
levels of 5-HT2C (Gurevich et al., 2002) and GluR2 receptors
(Burnashev et al., 1995). Furthermore, RNA editing can alter the sen-
sitivity and the pharmacological properties of channels and drug tar-
gets. For example, RNA editing of the Kv1.1 channels alters their sen-
sitivity to the Kv channel blocker 4-aminopyridine (4-AP) and reduce
the ictogenic potential to 4-AP in epileptic rats (Streit et al., 2011). In
addition, due to RNA editing in its α1 subunit, GABA receptor sensi-
tivity changes and its deactivation rate increases (Nimmich et al.,
2009).
To conclude, RNA editing can provide a selective effect on cell

19
N.S. Ganem et al.
sensitivity to various drugs by targeting relevant pathways and thus
should be considered in the development of novel therapeutics for
different indications.
Can A-to-I editing be used for cancer therapeutics?
Both ADARs and their edited transcripts are involved in cancer
progression. Therefore, ADARs might also be used as targets for novel
cancer therapies. Indeed, knocking out ADAR1 in a CML mouse model
caused rapid leukemic cell death (Steinman et al., 2013) and knocking
down ADAR1 in breast cancer cell lines caused increased apoptosis
(Fumagalli et al., 2015). ADAR1 is induced by the interferon response
pathway, which can be another possible therapeutic target (Patterson
and Samuel, 1995). However, ADARs have many roles in immunity and
organismal development, some of which are still unknown (Ganem and
Lamm, 2017; Goldstein et al., 2017). Therefore, targeting ADARs may
cause unforeseen and harmful side effects.
The discovery of the genome editing ability of the CRISPR-Cas9 tool
opened new avenues for successful gene therapies (Boettcher and
McManus, 2015; Cox et al., 2015; Fellmann et al., 2017). Although
other approaches such as delivery of shRNA are already on the market,
CRISPR-Cas9 DNA editing appears to be the most promising (Boettcher
and McManus, 2015). However, there are many obstacles that raise
concerns in using CRISPR-Cas9 for human therapies. Some of the con-
cerns are off-target editing in other DNA positions, the inability to ac-
cess the nucleus to reach genomic DNA, and the low efficiency of
editing (Cox et al., 2015).
In the last few years, several studies suggested some possible
modalities to exploit the capacity of ADAR proteins to edit RNA for
correcting genetic mutations on the mRNA levels or cause mutations in
specific positions (see below and in Fig. 3). The possibility to edit
mRNA instead of DNA is very appealing in terms of an enhanced ac-
cessibility to the mRNA, which resides in the cytoplasm, and a more
transient approach, which makes off-target editing less of a problem.
These new strategies utilize the ability of the ADAR enzymes to dea-
minate a certain nucleotide and convert it to another nucleotide. The
ability of ADAR enzymes to bind to their target substrates depends on
the RNA strand’s secondary structure. Altering the secondary structure
of the desired targets could enable the binding of ADAR enzymes and
consequent editing of the target. To date, a few site-directed RNA
editing (SDRE) techniques utilizing ADARs exist (for example: (Fukuda
et al., 2017; Montiel-Gonzalez et al., 2013; Stafforst and Schneider,
2012)). These techniques use different approaches to convert a single
nucleotide from adenosine to inosine. One approach (Stafforst and
Fig. 3. Site-directed RNA editing techniques. (A) ADAR1 deaminase domain (DD) fused to SNAP-tag protein which is conjugated to 5′-O-benzylguanine and guide RNA (gRNA). The
gRNA-target RNA complex undergoes RNA editing by the deaminase domain. (B) ADAR2 deaminase domain (DD) fused to a λ phage − N domain (λN-D). λN-D recognizes and binds a
boxB RNA, which is fused to a guide RNA (gRNA). The gRNA-target RNA complex undergoes RNA editing by the deaminase domain. (C) ADAR RNA binding domain (RBD) substrate
transcript fused to a guide RNA (gRNA) recruits the endogenous ADAR enzyme, which in turn binds it and deaminates the gRNA-target RNA complex.
20
Drug Resistance Updates 32 (2017) 16–22
Schneider, 2012) uses the ADAR1 deaminase domain without its RNA
binding domains (dsRBD) fused to a C-terminus SNAP-tag protein
conjugated to 5′-O-benzylguanine (BG) modified guide RNA (Fig. 3A).
The guide RNA recognizes its antisense target and forms a secondary
structure that enables editing by the ADAR1 deaminase domain. This
system was shown to successfully diminish a stop codon and correct
missense mutations in high efficiency in vitro and in vivo (Stafforst and
Schneider, 2012; Vogel et al., 2014), and was even adjusted to be light
activated (Hanswillemenke et al., 2015).
Another technique uses the ADAR2 deaminase domain fused to a λ
phage − N protein, a 22 amino acid peptide, which recognizes and
binds to the boxB RNA hairpin (Montiel-Gonzalez et al., 2013). To
target specific RNA, the boxB RNA is fused to an antisense guide RNA
that can bind to endogenous RNA (Fig. 3B). After recognizing the en-
dogenous RNA, it can undergo RNA editing by the ADAR2 deaminase
domain-λ-N fused protein. The system was shown to correct the W496X
mutation in the cystic fibrosis transmembrane conductance regulator
(CFTR) transcript both in vitro and in vivo (Montiel-Gonzalez et al.,
2013). While the percentage of corrected transcripts was very low,
CFTR activity was restored in vivo. The system’s efficiency was im-
proved by introducing an E488Q mutation in the deaminase domain, by
increasing the number of the boxB hairpins to two, and by using mul-
tiple λ-N peptides (Montiel-González et al., 2016).
A very recent approach has been to use a modified target RNA that
recruits endogenous ADARs and fuses them to an antisense fragment
complementary to the desired target (Fukuda et al., 2017; Heep et al.,
2017; Wettengel et al., 2017). The guide RNA is based on a known
ADAR substrate, which was shown to be efficiently edited. The anti-
sense fragment is designed as a template for the deamination. It as-
sembles to a dsRNA with the target RNA and undergoes RNA editing by
the ADAR deamination motif (Fig. 1C). Efficient target RNAs were
constructed to both ADAR1 isoforms and to ADAR2 (Fukuda et al.,
2017; Heep et al., 2017; Wettengel et al., 2017). This approach for site-
directed RNA editing might be more suitable for gene therapy and
mutation recoding since it uses the endogenous ADAR proteins and
does not require a newly constructed protein. This methodology was
used to correct a recessive stop mutation in PINK1, enabling mi-
tochondria autophagy in HeLa cells (Wettengel et al., 2017).
Although site-directed RNA editing technology is not as advanced as
CRISPR-Cas9 and was not tested yet in variety of disease models or
cancer cells, it bears a great potential. This is especially true if this
technique will pass the specific A-to-I editing barrier and will enable the
editing of other nucleotides.
N.S. Ganem et al.
Conclusions and future perspectives
Cancer studies involving ADAR RNA editing are the “new kids on
the block”. It is apparent that alterations in ADAR expression and
function in human malignancies affect the expression and activity of
many transcripts and consequently key proteins including oncogenes,
tumor suppressor genes and their regulators. It is still an open question
whether or not ADAR editing is a driver of cancer progression or simply
a passenger alteration. Nevertheless, RNA editing should be considered
as a biomarker for certain cancer types and stages as well as for ther-
apeutic drug interventions and outcomes. The most exciting aspect is
the possibility of using the ADAR mechanism for treating cancer and
other diseases. Transiently targeting specific RNA transcripts is very
appealing and with the development of targeted editing, this option
may become a realistic therapeutic approach in the future.
Acknowledgements
We thank Alla Fishman for critical reading of the manuscript and
discussions. This work was funded by The Israeli Centers of Research
Excellence (I-CORE) program, (Center No. 1796/12 to ATL), Israel
Cancer Research Fund (ICRF), and the Binational Israel-USA Science
Foundation (grant no. 2015091).
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