5. PHARMACOLOGICAL STRATEGIES AIM TO TARGET THE
EPITRANSCRIPTOME.
Taking into consideration the aforementioned dysregulation of the
epitranscriptome in cancer, the design and development of small
molecules that potentially revert defects in the epitranscriptome opens
new and exciting opportunities for drug discovery in oncology (Table 2).
Inevitabily, the successful introduction of epigenetic-based drugs into
the clinic delineates a model for chemical biology and drug discovery
research also on RNA-epitranscriptomics. Although the field of
epitranscriptome-targeting is still in its beginnings, preclinical
evidences of the benefits of RNA-modifying therapy are found in specific
experimental models. Most important, several biotech companies are
addressing the therapeutic potential of this promising field. Below, we
will highlight the emerging results and challenges for the use of RNA-
modifications as actionable targets for cancer drug discovery.
5.1. RNA methyltransferase inhibitors. There are several
parallelisms between DNA and RNA methylation that support their
exploitation within the framework of pharmacological inhibition. The
most similar chemical modification is the addition of a methyl group at
position 5 of cytidine resulting in 5-methylcytosine both at DNA or RNA
molecule. It is however not unreasonable to assume that current DNA
methyltransferases influence the dynamism of m5C RNA. As mentioned
before, drugs inhibiting DNA methylation (e.g., 5-Azacytidine (AZA) or
decitabine) have been FDA- approved and included in a clinical setting
for the treatment of haematological tumours (Berdasco and Esteller,
2018). However, it is known that the vast majority
(~90%) of AZA is incorporated into the RNA molecule and
that DNA methylation status does not correlate with the clinical
response to hypomethylating treatment. Whether the antiproliferative
effect is mediated by RNA or DNA methylation is still under debate and
further investigation is needed. Recently, a mechanism involving members
of the RNA methyltransferases NSUN (NSUN1 and NSUN3) together with DNMT2
has been proposed as a mediator of AZA response in a AML and
myelodysplasic syndrome model (Cheng et al., 2018). Specifically,
authors proposed a mode of action that involves the formation of two
chromatin complexes including distinct RNA modifiers to explain positive
response or resistance to AZA treatments. In AZA sensitive cells, the
reader hnRNPK directly recognized NSUN3, DNMT2 and CDK9/P-TEFb to
recruit RNA-pol-II \soutand resulting in an active conformation of
chromatin in sensitive AML cells. In AZA-resistant cells, the
interaction of NSUN1 with the chromatin remodelling factor BRD4 (but not
hnRNPK) and RNA pol-II results in an active chromatin structure that is
resistant to AZA. However, these AZA-resistant cells are sensitive to
the BRD4 inhibitor JQ1 and NSUN1 interference by siRNA (Cheng et al.,
2018). Supporting evidences on the impact of NSUN2 in AZA response
previously showed that NSUN2 and METTL1 abrogation (by genetic
knockdown) results in increased hypomethylating drug sensitivity in HeLa
cells (Okamoto et al., 2014). Since acquired resistance to chemotherapy
is a major bottleneck in cancer treatments, unravelling the multiple
molecular mechanisms that guides therapy response is a mandatory concern
to improve precision medicine in cancer.
Histone lysine methyltransferases (KMT) also contain SAM-binding pocket
and a substrate-binding domain that have been successfully targeted
(Ganesan et al., 2019). The first attempt to inhibit KMT activity was
based on the discovery that the natural product sinefungin reversibly
competes with SAM for its binding site (Kaniskan et al., 2015a).
Thereafter, several potent SAM-mimetics that are selective by taking
advantage of differences in the cofactor binding pocket have been
developed as KMT inhibitors. The DOT1L inhibitor, Pimenostat, was the
first KMT inhibitor to enter clinical trials for leukaemia therapy,
followed by EZH2 inhibitors (GSK2816126 and tazemetostat) approved for
B-Cell lymphoma treatments (Berdasco and Esteller, 2018). Although the
protein structure of specific RNA methyltransferases has specific and
unique features, like DOT1L, m5C and m6A RNA methyltransferases belong
to the Rossmann fold family of methyltransferases. This similarity
portends that KMT inhibitors could be used as a starting point for the
chemical design and drug development of RNA methyltransferases; however,
no strong preclinical data has been given to support this observation.
Considering that m6A is the most universal RNA modification together
with the well-defined aberrant m6A patterns associated with cancers, the
research community has already drawn attention to the importance of
strengthening the pharmacological manipulation of m6A methyltransferase
activity. METTL3-METTL4 is upregulated in cancers, and it has been
previously showed that genetic manipulation by CRISPR-Cas9 technology
guided to downregulate METTL3 enzyme prevent cell proliferation and
invasion in AML in vitro and in vivo models (Barbieri et
al., 2017). Consequently, drug developers from biotech companies have
started the race for early drug discovery targeting RNA
methyltransferases, mainly METTL3. Three companies, STORM Therapeutics
(Cambridge, UK), Accent Therapeutics (MA, USA) and Gotham Therapeutics
(NYC, USA), have announced to have METTL3 inhibitors in preclinical
phases ready for phase I clinical trials (Cully, 2019). To date, the
most advanced results have been achieved by STORM Therapeutics. In
October 2020, STORM \southas announced that its first-in-class drug
candidate targeting METTL3, named STC-15, has been selected to enter
human Phase I clinical trial as a therapy for refractory AML.
Preclinical studies on a mouse model of AML, showed that oral
administration of SCT-15 reduced both splenomegaly and the number of
circulating monocytes. Similarly, tumour growth was reduced in
patient-derived-xenografts (PDX)-AML models after treatment with the
METTL3 inhibitor (Cully, 2019). The company is now studying the
application on solid tumours. Accent Therapeutics has started its drug
discovery program with an initial investment of $40M to optimize the
selection of RNA-modifier inhibitors. They have announced that the
company has already found around 20 targets, with METTL3/METTL14
inhibitors for AML treatment at the front of their research
(Boriack-Sjodin et al., 2019; Cully, 2019). Similarly, Gotham
Therapeutics launched in October 2018 with a $54 million program is the
third company with a METTL3 inhibitor in preclinical development for AML
therapy (Cully, 2019).
Out of these few examples, discovery of METTL3-METTL14 inhibitors are
also explored in academia. Adenosine, one of the two moieties of SAM, is
a SAM-competitive inhibitor of METTL3 activity. Recently, starting from
a library of 4000 analogues and derivatives of the adenosine moiety of
SAM and using high-throughput docking into METTL3 and protein X-ray
crystallography, an adenosine derivative showed low μM potency and good
ligand efficiency (Bedi et al., 2020). Interestingly, authors showed
that the ribose of adenosine can be replaced by other ring systems,
opening new opportunities for additional modifications (Bedi et al.,
2020). Further development in preclinical models is still needed to
explore the biological effect and mode of action of these adenosine
derivatives.
5.2. RNA demethylases inhibitors. RNA demethylases also
exhibit structural similarities with the protein lysine demethylases
from the Jumonji C (JMJC) family. These are part of the 2-oxoglutarate
and iron (II)-dependent dioxygenase family. This similarity is very
interesting given that current inhibitors of JMJC proteins are available
(Hauser et al., 2018) and that the mechanistic similarity between JMJC
and RNA demethylases could facilitate the drug discovery for the
inhibition of RNA modifications.
Interestingly, RNA demethylases have been targeted by specific
small-molecule inhibitors. As m6A dysregulation impact in normal
development and disease, its inhibition has been in the spotlight in the
last years (Table 2 ). A pioneer study of small-molecule
inhibitors of the human FTO demethylase was achieved by an chemical
optimization of the natural product rhein (Chen et al., 2012). Rhein
competitively binds to the FTO active site in vitro and globally
increases cellular m6A on mRNA in the BE-2(C) cell line (Chen et al.,
2012). Rhein also binds to ALKBH2 and ALKBH3 m1A and m3C demethylase,
respectively; however, different binding sites are involved for ALKB or
FTO inhibition (Li et al., 2016a).
A selective inhibition of m6A demethylase FTO rather than ALKBH5 was
reported (Huang et al., 2015). The work was based on the identification
of the differences in the displacement of
m6A-containing ssDNA binding to FTO and ALKBH5. This
screening provides meclofenamic acid (MA), previously identified as an
anti-inflammatory, as the best match to specifically inhibit FTO over
ALKBH5 (Huang et al., 2015). In vitro studies demonstrated that
treatment of HeLa cancer cells with the ethyl ester form of MA (MA2)
increased m6A mRNA levels (Huang et al., 2015). Furthermore, the
antiproliferative effect of MA2 treatment has been tested in in
vivo models of glioblastoma (Cui et al., 2017). MA2 increased mRNA m6A
levels in glioblastoma-stem cells (GSC) leading to suppression of the
GSC-initiated brain tumour development and prolonged the lifespan of
GSC-engrafted mice (Cui et al., 2017). This result suggests that
targeting m6A methylation could be a promising strategy for the
treatment of glioblastoma. Using leukaemia in vitro and in
vivo models, a role for the pharmacological inhibition of FTO to
prevent resistance to tyrosine kinase inhibitor (TKI) therapy has been
demonstrated (Yan et al., 2018). Mechanistically, exposure to rhein or
MA increases m6A and mRNA stability of survival and proliferation genes
(e.g. BCL-2 or MERTK) improving the sensitivity of the tumour to the TKI
nilotinib and PKC412 (Yan et al., 2018).
The knowledge gained on the basis for MA selectivity for FTO over ALKBH5
has facilitated the design of additional FTO inhibitors. In a later
step, a study based on a screening of many fluorescent molecules with
structures similar to MA revealed that fluorescein (and some of its
derivatives) selectively inhibited FTO demethylation as well as directly
labelled FTO protein (Wang et al., 2015). Two fluorescein derivatives
with improved cell permeability, FL6 and FL8, could efficiently inhibit
FTO demethylation and modulate the level of m6A in the mRNA of living
cells (Wang et al., 2015).
Research aimed at developing selective and cell-active small molecule
inhibitors of AlkB subfamilies of demethylases have also explored the
nucleotide-binding site instead of the 2OG-binding site (Toh et al.,
2015). Compound 12 exhibits 30-fold to 130-fold selectivity for FTO over
other AlkB subfamilies, and what is probably more interesting, the
compound also discriminates against other human 2OG oxygenases, as PHD2
and JMJD2A protein demethylases. Treatment with an ethyl ester
derivative of compound 12 increases m6A in HeLa cells (Toh et al.,
2015).
Using structure-based rational design, which maintains the benzyl
carboxylic acid to keep MA selectivity for FTO but extends the
dichloride-substituted benzene to a deeper pocket that could be fully
occupied by a bulky ligand, two FTO inhibitors were developed (FB23 and
FB23-2) (Huang et al., 2019c). Based on the discovery that FB23 showed
increased m6A levels due to FTO inhibition in in vitro AML cell
lines, researchers have optimized the physicochemical property of FB23
and produced FB23-2 compound with a significantly improved antitumoural
ability in in vitro but also in vivo conditions. FB23-2
treatments reduces the proliferation of a panel of AML cell lines, but
most importantly, FB23-2 also inhibits primary leukaemia stem cells in
PDX –AML mice models. Mechanistically, gain of m6A levels after FB23-2
treatment modulates mRNA transcripts associated with proliferation
(e.g., MYC, CEBPA, RARA, and ASB2) (Huang et al., 2019c).
Additional FTO inhibitors were discovered using an elegant
high-throughput screen using the fluorogenic methylated Broccoli
substrate HTS assay (Svensen and Jaffrey, 2016). These Broccoli assays
are based on the construction of a fluorescent RNA-dye complex that
appear non-fluorescent when it contains m6A but becomes fluorescent
after demethylation. The study identified several selective compounds
for FTO inhibition which increase m6A levels at FTO target mRNAs (bone
morphogenetic protein 6 (BMP6) and ubiquitin C (UBC) in HEK293C cells
(Svensen and Jaffrey, 2016).
The possibilities extend beyond rational drug design as we learn more of
the mode of action of the m6A-FTO axis. For example, the oncometabolite
R–2-hydroxyglutarate (R-2HG) is produced at high levels in mutant
isocitrate dehydrogenase 1/2 (IDH1/2) leukaemia cells (Su et al., 2018).
However, R-2HG also has an antitumoral effect through the inhibition of
FTO activity. FTO inhibition results in a gain of m6A levels and the
stabilization of the mRNA transcripts MYC/CEBPA, leading to the
suppression of relevant proliferation pathways (Su et al., 2018).
Undoubtedly, the primary focus of attention is now in FTO inhibition.
However, additional RNA demethylases could be “druggable” targets. For
example, the small compound
1-(5-methyl-1H-benzimidazol-2-yl)-4-benzyl-3- methylpyrazol-5-ol
(HUHS015) was able to inhibit the prostate cancer antigen-1
(PCA-1/ALKBH3) axis in prostate cancer cell lines and murine xenograft
models for prostate cancer (Nakao et al., 2014).
5.3. Targeting other RNA modifications. Epigenetic
modifications of DNA molecules are interpreted by a set of reader
proteins with essential functions. Most importantly, these readers for
epigenetic marks are altered in human diseases, leading to the discovery
of small compound inhibitors of their activity (Ganesan et al., 2019).
Potent drug inhibitors have been identified for the H3K27me3 reader
Polycomb protein EED from the Polycomb repressive complex 2 (PRC2)
family (He et al., 2017). Like The YTF family of RNA methyl readers,
Polycomb protein EED contains an aromatic cage crucial for the
recognition of the methyl group. Whether the success of drug discovery
associated with methyl-lysine readers could be translated to the
methyl-RNA-reader field is still uncertain and unexplored. One barrier
to the development of YTF- inhibitors is that YTF members from the same
family of proteins exhibit high structural homology. It still needs to
be determined whether the application of Pan-YTF inhibitors could have a
tissue-specific effect and contribute to increased specificity (Cully,
2019). Efforts to inhibit the m7G reader eukaryotic translation
initiation factor 4E (eIF4E) as an actionable target have been proposed
(Soukarieh et al., 2016). Through its role in the regulation of mRNA
translation of oncogenic pathways, eIF4E is implicated in cell
transformation, tumorigenesis, and angiogenesis. Guanine-based
inhibitors of eIF4E were evaluated in in vitro cell-based assays
and provides a set of compounds with inhibitory activity at
physiological doses (Soukarieh et al., 2016).
There are unique RNA modifications that could not be compared with DNA
or histone modifications. The RNA modifications involved in
pseudouridylation or A-to-I editing have no precedents in drug
discovery. However, chemical biology and drug discovery in this area
requires a better characterization of the modes of action and
pathological implications in a context-specific manner. The 3’ terminal
uridylyl transferase Zcchc11 (TUT4) is recruited to precursor let-7 RNA
to selectively block let-7 miRNA biogenesis, a miRNA with tumour
suppressor properties. Downregulation of Let7 miRNA has been described
in cancer. It is therefore of great application to develop an inhibitor
for the uridylation of precursor let-7 resulting in restored Let-7
expression in cancer (Lin and Gregory, 2015). Using an automated
high-throughput screen of ∼15,000 chemicals, some small compounds have
been selected as putative TUTase inhibitors (Lin and Gregory, 2015). The
understanding of the TUT4-let-7 mediated inhibition is not addressed, so
the consequences in preclinical models need to be determined.
In terms of pharmacological inhibition of the A-to-I edition, targeting
ADAR family of proteins could be a promising strategy for cancer
therapy. As mentioned before, ADAR1 is involved in multiple
cancer-related pathways, and loss of function of ADAR1 in tumour cells
profoundly sensitizes tumours to anti-PD1 immunotherapy (Ishizuka et
al., 2019). Consequently, a strategy to repress ADAR1 expression is
particularly challenging. Nowadays, there is not any public compound
targeting ADAR1; but biotech companies such as Accent Therapeutics have
declared to be working on this target for NSCLC therapy (Cully, 2019).
In addition, by studying analogues of a naturally adenosine analogue,
the 8-azaadenosine compound showed in vitro inhibition activity
of ADAR2 (Véliz et al., 2003).