Modulation of RNA splicing as a potential treatment for cancer

Article (PDF Available)inBioengineered bugs 2(3):125-8 · May 2011with29 Reads
DOI: 10.4161/bbug.2.3.15165 · Source: PubMed
Abstract
Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs. SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ∆7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.
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Bioengineered Bugs 2:3, 125-128; May/June 2011; © 2011 Landes Bioscience
COMMENTARYCOMMENTARY
Key words: cancer, antisense,
oligonucleotide, splicing, apoptosis
Submitted: 01/26/11
Revised: 02/12/11
Accepted: 02/15/11
DOI: 10.4161/bbug.2.3.15165
*Correspondence to: John A. Bauman;
Email: bau@email.unc.edu
C
lose to 90% of human genes are
transcribed into pre-mRNA that
undergoes alternative splicing, pro-
ducing multiple mRNAs and pro-
teins from single genes. This process is
largely responsible for human proteome
diversity, and about half of genetic
disease-causing mutations affect splic-
ing. Splice-switching oligonucleotides
(SSOs) comprise an emerging class of
antisense therapeutics that modify gene
expression by directing pre-mRNA splice
site usage. Bauman et al. investigated an
SSO that upregulated the expression of
an anti-cancer splice variant while simul-
taneously eliminating an overexpressed
cancer-causing splice variant. This was
accomplished by targeting pre-mRNA
of the apoptotic regulator Bcl-x, which
is alternatively spliced to express anti-
and pro-apoptotic splice variants Bcl-x
L
and Bcl-x
S
, respectively. High expression
of Bcl-x
L
is a hallmark of many cancers
and is considered a general mechanism
used by cancer cells to evade apoptosis.
Redirection of Bcl-x pre-mRNA splic-
ing from Bcl-x
L
to -x
S
by SSO induced
apoptotic and chemosensitizing effects
in various cancer cell lines. Importantly,
the paper shows that delivery of Bcl-x
SSO using a lipid nanoparticle redirected
Bcl-x splicing and reduced tumor burden
in melanoma lung metastases. This was
the first demonstration of SSO efficacy
in tumors in vivo. SSOs are not limited
to be solely potential anti-cancer drugs.
SSOs were first applied to repair aber-
rant splicing in thalassemia, a genetic
disease, they have been used to create
novel proteins (e.g., Δ7TNFR1), and
they have recently progressed to clinical
trials for patients with Duchenne muscu-
lar dystrophy.
Modulation of RNA splicing as a potential treatment for cancer
John A. Bauman
1,
* and Ryszard Kole
1,2
1
Department of Pharmacology; University of North Carolina; Chapel Hill, NC USA;
2
AVI Biopharma Inc.; Bothell, WA USA
Pre-mRNA splicing, the co-transcrip-
tional process of intron removal and exon
joining seen in higher eukaryotes, was first
thought to be an exception to the rule that
mRNAs are carbon copies of their respec-
tive genes. Sequencing of the human
genome revealed that not only splicing but
also alternative splicing, which enables a
single pre-mRNA transcript to give rise
to multiple distinct splicing isoforms and
therefore multiple variant proteins, are
the norm. Alternative splicing explains
how the relatively limited human genome
(~30,000 genes) gives rise to more than
100,000 proteins. Recent analyses indi-
cate that nearly 95% of all multi-exon
gene transcripts undergo alternative splic-
ing, 86% with a minor isoform frequency
of 15% or more.
1
In addition to regulating
gene expression, pre-mRNA splicing and
alternative splicing are being recognized
for their roles in human disease. Indeed,
up to half of disease-causing genetic muta-
tions affect pre-mRNA splicing.
2
The
near-ubiquity of alternative splicing and
the prevalence of splicing aberrations in
human disease open up a new field that
can result in the harvest of new gene- and
sequence-specific medicines that achieve
clinical benefits by modulating splicing.
The SSO described in the Bauman et al.
paper has the potential to be one such
medication.
Splice-Switching Oligonucleotide
Mechanism of Action
and Applications
Splice-switching oligonucleotides (SSOs)
comprise an emerging class of antisense
drug candidates that modify pre-mRNA
splicing. They are designed to block
sequences in pre-mRNA essential for
©2011 Landes Bioscience.
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126 Bioengineered Bugs Volume 2 Issue 3
has been detected in a number of malig-
nancies. A recent study of 3,131 cancer
samples across more than two dozen can-
cer types found that the bcl- x gene was
amplified in almost all of them, suggest-
ing that upregulation of this gene may be
a common mechanism by which cancers
increase survival.
17
Previously, this labora-
tory targeted Bcl-x in prostate and breast
cancer cells,
18,19
whereas the work dis-
cussed here focused on Bcl-x in metastatic
melanoma.
14
Use of the upstream 5' alternative splice
site of Bcl-x exon 2 produces an internally
deleted protein, Bcl-x
S
(Fig. 1).
15
Bcl-x
S
is
anti-apoptotic because it directly binds
and inhibits the pro-apoptotic Bcl-x
L
and
Bcl-2 proteins.
20
The authors show that
targeting the downstream (Bcl-x
L
) splice
site with a 2'MOE SSO redirects the splic-
ing machinery to the upstream (Bcl-x
S
)
splice site, effectively converting an anti-
apoptotic molecule into a pro-apoptotic
molecule. As a result, the tumor load in
treated mice is reduced. Previously it
was shown that newly generated Bcl-x
S
induced by SSO also sensitizes cancer cells
to treatment with UV- and γ-irradiation
and chemotherapeutic drugs, including
etoposide, 5-fluorouracil, cisplatin, 5-fluo-
rodeoxyuridine and doxorubicin.
18,21
Two significant characteristics of splic-
ing modulation that distinguish this
approach from downregulation of anti-
apoptotic genes by ASOs and siRNA
are worth highlighting.
22
First, in every
instance of SSO-redirected splicing of
Bcl-x pre-mRNA from Bcl-x
L
to Bcl-x
S
an
anti-apoptotic Bcl-x
L
molecule is elimi-
nated and the pro-apoptotic Bcl-x
S
mol-
ecule appears. The latter, as mentioned
above, will inhibit existing Bcl-x
L
and
Bcl-2 proteins, further reducing resistance
of cancer cells to chemotherapy and apop-
tosis. The SSO thus produces a double or
triple bang for the buck.
Second, the higher the level of expres-
sion of Bcl-x pre-mRNA in a cell, the more
SSO-induced, anti-apoptotic Bcl-x
S
mole-
cules that will be produced. This suggests
that cells from aggressive cancers with
higher levels of Bcl-x expression will be
more susceptible to SSO-induced apopto-
sis than healthy, untransformed cells. This
counterintuitive mechanism should thus
reduce the undesirable side effects that
cells is their ability to evade and corrupt
apoptosis, prompting the search for drugs
that can restore or potentiate apoptotic
signaling in tumor cells.
10
Apoptosis is regulated by interactions
among members of the Bcl-2 family of
proteins, some of which (e.g., Bax and
Bak) are pro-apoptotic and induce pro-
grammed cell death. Some are anti-apop-
totic (e.g., Bcl-2 and Bcl-x
L
), and prevent
the cell from committing suicide unneces-
sarily. A fine balance is maintained until
an apoptotic event, such as DNA damage
or growth factor deprivation, prompt Bax
and Bak to trigger apoptosis.
11,12
However,
if anti-apoptotic Bcl-2 and/or Bcl-x
L
pro-
teins are overexpressed they can overcome
Bax and Bak, enabling the cell to survive
with damaged or rearranged DNA and
in the absence of growth factors. The cell
is converted into a cancer cell that is free
to proliferate, escaping normal controls.
Because many chemotherapeutics induce
apoptosis through Bax and Bak-like pro-
teins, cancer cells with excess of Bcl-2
and Bcl-x
L
become resistant to chemo-
therapy.
13
It follows that a drug that can
reactivate the apoptotic pathway could be
a good anti-cancer agent in its own right
and/or could re-sensitize the cancer cell to
chemotherapy.
Bcl-x undergoes splicing at two alterna-
tive 5’ splice sites of exon 2 (Fig. 1), yielding
two protein with opposing functions, Bcl-x
L
and -x
S
.
15
Use of the downstream splice
site produces Bcl-x
L
, which exerts an
anti-apoptotic effect by antagonizing
and inhibiting the pro-apoptotic Bax and
Bak proteins. It also induces growth of
blood vessels that vascularize the tumor.
16
Not surprisingly, Bcl-x
L
overexpression
splicing, redirecting the splicing machin-
ery to a new pathway. That this mecha-
nism is feasible in vitro, in vivo in animals,
and in patients has been demonstrated in
numerous publications from this labora-
tory and by others.
3-7
The SSO mechanism of action is dis-
tinct from conventional antisense oligonu-
cleotides (ASOs) and siRNAs that inhibit
gene expression by degrading the target
mRNA through RNase H and the RISC
complex, respectively. Instead, SSOs ste-
rically block sequences in pre-mRNA
without leading to their degradation,
thereby shifting the pattern of splicing.
This action requires that the SSO form
very stable duplexes with its pre-mRNA
target to enable successful redirection of
the splicing factors to alternative, desired
sequences. These requirements are met
by incorporating various chemical modi-
fications to the oligonucleotide backbone
that improve binding afnity and do not
support RNase H cleavage. Synthetic
SSO chemistries include 2'-O-methyl,
2'-O-methoxyethyl (2'MOE), phospho-
rodiamidate morpholino, peptide nucleic
acid and locked nucleic acid.
8,9
Splice-Switching as a Potential
Cancer Therapy
Defective human cells can be pushed onto
a suicide pathway through a programmed
cell death mechanism called apoptosis.
Apoptosis is essential for normal develop-
ment and maintenance of tissue homeo-
stasis; however, dysregulation of apoptotic
signaling contributes to numerous patho-
logical conditions, including cancer.
Indeed, one of the hallmarks of cancer
Figure 1. Splicing at the downstream or upstream 5' alternative splice site of Bcl-x exon two pro-
duces Bcl-x
L
or Bcl-x
S
, respectively. Bcl-x
L
is highly expressed in many human cancers and confers
resistance to a broad range of chemotherapeutic agents. It has also been shown to play a role in
tumor angiogenesis. Bcl-x
S
is a pro-apoptotic protein capable of binding and antagonizing Bcl-2
and Bcl-x
L
. SSO targeted to the downstream 5' alternative splice site switches production from
Bcl-x
L
to Bcl-x
S
, inducing apoptosis and chemosensitization.
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SSO only or nanoparticles formulated
with a control SSO. The paper presents
the first demonstration of sequence-spe-
cific SSO efficacy in tumors in vivo.
Table 1 shows other potential targets for
SSOs as anti-cancer drugs, indicating that
this approach is not limited to the Bcl-x
gene alone.
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Cancers with high Bcl-x
L
expression and
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19
In contrast, RNase H-sensitive
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Table 1. Examples of genes involved in the proliferation, survival and chemoresistance of cancer cells that express splice variants with different
functions (reviewed in refs. 26–28). This non-comprehensive list suggests a richness of potential SSO targets for cancer therapy.
Role in cancer Gene Splice variants Cellular effect
Apoptosis Bcl-x Bcl-x
L
Anti-apoptotic, promotes chemoresistance
Bcl-x
S
Pro-apoptotic, antagonizes Bcl-x
L
and Bcl-2
Mcl-1 Mcl-1
L
Anti-apoptotic, promotes chemoresistance
Mcl-1
S
Pro-apoptotic, antagonizes Mcl-1
L
Caspase-2 Caspase-2
L
Pro-apoptotic
Caspase-2
S
Anti-apoptotic, protects against chemotherapeutics
Caspase-9 Caspase-9 Pro-apoptotic
Caspase-9B Anti-apoptotic, inhibits apoptosome formation
Survivin Survivin Anti-apoptotic
Survivin-2B Pro-apoptotic, antagonizes survivin
Cell proliferation Fas Fas Mediates apoptotic signaling
FasExo8Del Inhibits Fas-mediated apoptosis, upregulated in certain cancers
HER2 HER2 Promotes proliferation and survival of cancer cells
Herstatin Pro-apoptotic, soluble dominant-negative inhibitor of HER2
Δ15HER2
Pro-apoptotic, soluble dominant-negative inhibitor of HER2
Rac1 Rac1 Regulates cell proliferation and cytoskeletal reorganization
Rac1b
Increased rate of GDP/GTP exchange leads to constitutive activation,
transforms cells in culture, expressed exclusively in tumor tissue
Angiogenesis VEGF VEGFA
Promotes angiogenesis through activation of VEGF receptors 1 and 2,
upregulated in many cancers
VEGF165b Inhibits angiogenesis through competitive inhibition of VEGF receptor 2
Transcription factors p53 p53 Tumor suppressor, transcription factor
p47 Antagonizes p53 tumor suppressor
KLF6 KLF6 Tumor suppressor, transcription factor
KLF6-SV1 Antagonizes KLF6, upregulated in certain cancers
©2011 Landes Bioscience.
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128 Bioengineered Bugs Volume 2 Issue 3
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    • "However, the demonstration that HBx can interact with splicing factor SF1 is clearly a step towards identifying the steps required for the extensive modifications of AS observed in HCC. Interestingly, strategies to modulate AS by splice-switching oligonucleotides in order to correct aberrant ASEs, and/or to induce expression of therapeutic splice variants are now being developed [17, 54]. It is tempting to speculate that such a strategy could be applied to HCC. "
    [Show abstract] [Hide abstract] ABSTRACT: Background Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. ResultsOur study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. Conclusions This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.
    Full-text · Article · Dec 2016
    • "Strategies to modulate AS by splice-switching oligonucleotides in order to correct aberrant events, or to induce expression of therapeutic splice variants are being developed [17,20,21] . For instance, the splicing of Bcl-x(L) in cancer cells can be redirected towards the pro-apoptotic variant Bcl-x(S), which has been shown to reduce the tumor load in xenografts of metastatic melanoma [70]. It is therefore tempting to speculate that such a strategy could likely be utilized to limit viral replication. "
    [Show abstract] [Hide abstract] ABSTRACT: Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.
    Full-text · Article · Sep 2016
    • "The use of the upstream 5 alternative splice site of bcl-x exon 2 produces Bcl-xS, which directly inhibits the pro-apoptotic Bcl-xL and Bcl-2 proteins. Targeting the downstream (bcl-xL) splice site with a 2 -O-Me redirects the splicing machinery to the upstream (bcl-xS) splice site, converting it into a pro-apoptotic molecule to reduce tumor growth [14]. Considering that up to 50% of human disease-causing mutations affect splicing, the SSO approach is emerging as a promising alternative to gene therapy. "
    [Show abstract] [Hide abstract] ABSTRACT: Peptides are versatile and attractive biomolecules that can be applied to modulate genetic mechanisms like alternative splicing. In this process, a single transcript yields different mature RNAs leading to the production of protein isoforms with diverse or even antagonistic functions. During splicing events, errors can be caused either by mutations present in the genome or by defects or imbalances in regulatory protein factors. In any case, defects in alternative splicing have been related to several genetic diseases including muscular dystrophy, Alzheimer's disease and cancer from almost every origin. One of the most effective approaches to redirect alternative splicing events has been to attach cell-penetrating peptides to oligonucleotides that can modulate a single splicing event and restore correct gene expression. Here, we summarize how natural existing and bioengineered peptides have been applied over the last few years to regulate alternative splicing and genetic expression. Under different genetic and cellular backgrounds, peptides have been shown to function as potent vehicles for splice correction, and their therapeutic benefits have reached clinical trials and patenting stages, emphasizing the use of regulatory peptides as an exciting therapeutic tool for the treatment of different genetic diseases. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Mar 2015
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