Biological validation that SF3b is a target of the antitumor
macrolide pladienolide
Akira Yokoi, Yoshihiko Kotake, Kentaro Takahashi, Tadashi Kadowaki, Yoshiko Matsumoto,
Yukinori Minoshima, Naoko H. Sugi, Koji Sagane, Makoto Hamaguchi, Masao Iwata and
Yoshiharu Mizui
Tsukuba Research Laboratories, Eisai Co., Ltd, Tsukuba, Ibaraki, Japan
Keywords
antitumor activity; pladienolide; resistant
mutation; SF3b; splicing inhibitor
Correspondence
A. Yokoi, Eisai Co., Ltd, Tokodai 5-1-3,
Tsukuba, Ibaraki 300-2635, Japan
Fax: +81 29 847 2759
Tel: +81 29 847 5808
E-mail: a-yokoi@hhc.eisai.co.jp
(Received 21 July 2011, revised
13 September 2011, accepted 26
September 2011)
doi:10.1111/j.1742-4658.2011.08387.x
Pladienolide is a naturally occurring macrolide that binds to the SF3b com-
plex to inhibit mRNA splicing. It has not been fully validated whether the
splicing impairment is a relevant mechanism for the potent antitumor activ-
ity of pladienolide. We established pladienolide-resistant clones from WiDr
and DLD1 colorectal cancer cells that were insensitive to the inhibitory
action of pladienolide on cell proliferation and splicing. An mRNA-Seq
differential analysis revealed that these two cell lines have an identical
mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of
the SF3b complex. Reverse expression of the mutant protein transferred
pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation
analysis using a radiolabeled probe showed that the mutation impaired the
binding affinity of paldienolide to its target. These results clearly demon-
strate that pladienolide exerts its potent activity by targeting SF3b and also
suggest that inhibition of SF3b is a promising drug target for anticancer
therapy.
Structured digital abstract
lSF3B1 physically interacts with SF3B2 and SF3B3 by anti bait coimmunoprecipitation (View
interaction)
Introduction
Pladienolide is a 12-membered macrolide that was first
isolated from Streptomyces platensis Mer-11107 during
a cell-based assay that evaluated the suppression of
hypoxia-induced gene expression controlled by the
human vascular endothelial growth factor promoter
[1,2]. Pladienolide B has been shown to arrest cell-cycle
progression during the G1 phase and the G2 ⁄M transi-
tion of the cell cycle in vitro, and also to inhibit tumor
growth in several human cancer xenograft models in
mice [3]. COMPARE analysis with panel screening of
39 human cancer cell lines indicated that pladienolide
B has a unique mode of antitumor action, unlike that
of the anticancer drugs currently in clinical use [4].
Photoaffinity-labeling studies identified splicing factor
SF3b as a major binding target molecule for pladieno-
lide [5]. SF3b is a key component of the U2 small
nuclear ribonucleoprotein (snRNP) complex that is
responsible for the splicing of precursor messenger
RNA (pre-mRNA) and the formation of mature
mRNA. Recently, E7107, a synthetic derivative of pla-
dienolide D, was shown to prevent the tight binding of
U2 snRNP to pre-mRNA, resulting in the formation
of defective spliceosomes. E7107 was also shown to
impair an ATP-dependent remodeling event in U2
snRNP that exposes the branchpoint binding
region [6].
Abbreviations
HA, hemagglutinin; Ig, immunoglobulin; pre-mRNA, precursor messenger RNA; snRNP, small nuclear ribonucleoprotein.
4870 FEBS Journal 278 (2011) 4870–4880 ª2011 The Authors Journal compilation ª2011 FEBS
SF3b is a 450 kDa complex that comprises seven su-
bunits: SF3B1, SF3B2, SF3B3, SF3B4, SF3B5,
SF3B14 and PHF5A [7–10]. At 155 kDa, SF3B1 is the
largest subunit, and it cross-links to pre-mRNA both
5¢and 3¢of the branchpoint [11]. The N-terminal 450
amino acid region of SF3B1 functions as a scaffold to
facilitate interaction with other splicing factors such as
U2AF65 and SF3B14 [12]. The C-terminal region of
SF3B1 contains 22 tandem repeats of the HEAT motif
[13]. A single HEAT motif consists of approximately
40 amino acids forming two antiparallel a-helices, and
tandem repeats of the motif are present in a variety of
proteins, such as protein phosphatase 2A, importin-b
and eukaryotic initiation factor 4G [14–16]. The
HEAT repeats of SF3B1 meander around the SF3b
complex, enclosing SF3B14 [17].
Although the binding affinities of pladienolide deriv-
atives to SF3b correlate with their inhibition of cell
proliferation [5], the precise antitumor mechanism of
the compounds has not been fully elucidated. In the
present study, we show that a R1074H mutation in the
gene for SF3B1 confers resistance to the inhibitory
action of pladienolide on cell proliferation and splicing
by impairing the ability of pladienolide to bind to the
SF3b complex. We also present evidence that the tar-
geting of SF3b is the direct mechanism for the anti-
tumor activity of pladienolide.
Results
Establishment of pladienolide-resistant cells
To elucidate the antitumor mechanism of pladienolide,
we established pladienolide-resistant (-R) cells using
WiDr and DLD1 human colorectal cancer cell lines.
WiDr-R and DLD1-R clones were obtained from their
parental cells by stepwise selection with increasing con-
centrations of pladienolide B and E7107, respectively,
followed by limiting dilution cloning. Pladienolide B
showed potent antiproliferative action against the
parental WiDr and DLD1 cells, with IC
50
values of
0.5 and 8.5 nM, respectively; however, WiDr-R and
DLD1-R cells continued to proliferate even when trea-
ted with 100 nMpladienolide B (Fig. 1B). We also
confirmed that WiDr-R and DLD1-R cells exerted
cross-resistance against E7107 (Fig. S1). To further
characterize the resistant cell lines, we assessed the pla-
dienolide-induced inhibition of splicing. After 4 h of
exposure to pladienolide B, the amounts of unspliced
mRNA of DNAJB1, CDKN1B, RIOK3 and BRD2
were measured using quantitative PCR analysis. Pladi-
enolide B significantly increased the unspliced forms of
the mRNA in WiDr and DLD1 cells in a dose-depen-
dent manner. However, accumulation of the unspliced
mRNAs was not observed in WiDr-R and DLD1-R
cells treated with pladienolide B (Fig. 2). These results
demonstrate that the WiDr-R and DLD1-R cells are
resistant to both the cell growth suppression and splic-
ing inhibition of pladienolide.
Identification of the SF3B1 mutation in
pladienolide-resistant cells
To examine the mechanism of resistance in WiDr-R
and DLD1-R cells, we performed mRNA-Seq differen-
tial analysis using the Illumina Genetic Analyzer (Illu-
mina, San Diego, CA, USA). Deep sequencing was
used to compare mRNA sequences in paired parental
and resistant WiDr and DLD1 cells. The two sets of
analyses generated 98 and 83 Mb sequences, respec-
tively, which each aligned with the human reference
sequence, and identified four and 87 mutations specific
to the resistant lines in WiDr ⁄WiDr-R and
DLD1 ⁄DLD1-R pairs, respectively (Fig. 3A). One muta-
tion in the gene for SF3B1 that results in the replace-
ment of Arg1074 with a His was common between the
two resistant clones. The R1074H mutation was con-
firmed using the Sanger sequencing method, which
demonstrated that the mutant mRNA was heterozy-
gously expressed in WiDr-R cells but was homozy-
gously expressed in DLD1-R cells (Fig. 3B).
The SF3B1 mutation confers pladienolide
resistance
To investigate whether the R1074H mutation in the
gene for SF3B1 directly confers pladienolide resistance
to cells, we performed a reverse-expression analysis of
the mutant protein using parental WiDr cells. Accord-
ingly, two types of viral vectors were prepared: (a) an
SF3B1 expression vector, which expressed wild-type or
R1074H-type hemagglutinin (HA)-tagged SF3B1, and
(b) an SF3B1 knockdown vector, which targeted the
3¢-UTR sequences of SF3B1 to decrease the endo-
genous expression of the gene (Fig. 4A). After trans-
duction of both expression and knockdown viral
vectors into WiDr cells, we confirmed the expression
of the HA-tagged SF3B1 protein by western blot anal-
ysis using anti-HA serum (Fig. 4B). Knockdown of the
endogenous SF3B1 expression was also evaluated by
quantitative PCR analysis using primers designed for
the SF3B1 3¢-UTR (Fig. 4C), and the results obtained
showed that most of the endogenous SF3B1 protein
was replaced with the exogenous HA-tagged SF3B1.
The effect of pladienolide B on the cell growth of these
transduced WiDr cells was investigated. Pladienolide B
A. Yokoi et al. SF3b as an antitumor target of pladienolide
FEBS Journal 278 (2011) 4870–4880 ª2011 The Authors Journal compilation ª2011 FEBS 4871
showed potent action with IC
50
values of 0.8 nMin
wild-type SF3B1 expressed ⁄endogenous SF3B1-knock-
down cells (Fig. 5A and Fig. S2A). This value was
almost identical to that in nontransduced WiDr cells
(Fig. 1B), demonstrating that exogenous expression of
SF3B1 did not affect the sensitivity to pladienolide B.
O
O
O
OH
O
OH
O
R1
R2
7
OH
CH3
CH3
C2H5NH
4-cycloheptylpiperazin-1-yl
Pladienolide B
3H probe
Pladienolide D
E7107
H
H
OH
OH
Compound R1R2
A
0
20
40
60
80
100
120
0.1
0.01 110100
Pladienolide B (nM) Pladienolide B (nM)
Cell viability (% of control)
WiDr
WiDr-R
WiDr
0
20
40
60
80
100
120
0.01 0.1 1 10 100
Cell viability (% of control)
DLD1
DLD1-R
DLD1
B
Fig. 1. Establishment of pladienolide-resistant cell lines. (A) Structures of pladienolides B and D,
3
H probe and E7107. (B) Antiproliferative
action of pladienolide B against WiDr ⁄WiDr-R and DLD1 ⁄DLD1-R cells. Cell viability was assessed with a colorimetric assay after 72 h of
treatment with pladienolide B. Each data point represents the mean ± SD from three separate experiments, each performed in triplicate.
0
20
40
60
80
100
120
0 1 10 100
Pladienolide B (nM)
Unspliced mRNA (fold change)
WiDr
WiDr-R
0
2
4
6
8
10
12
14
16
0110100
Pladienolide B (nM)
WiDr
WiDr-R
0
1
2
3
4
5
6
7
8
9
10
0110100
Pladienolide B (nM)
0
2
4
6
8
10
12
14
16
0 1 10 100
Pladienolide B (nM)
DLD1
DLD1-R
0
2
4
6
8
10
12
14
16
0110100
Pladienolide B (nM)
0
5
10
15
20
25
0 1 10 100
Pladienolide B (nM)
Unspliced mRNA (fold change)
DLD1
DLD1-R
A
B
WiDr
WiDr-R
DLD1
DLD1-R
DNAJB1 CDKN1B
DNAJB1 CDKN1B RIOK3
BRD2
Fig. 2. Splicing inhibition by pladienolide B. (A) WiDr ⁄WiDr-R cells and (B) DLD1 ⁄DLD1-R cells were treated with 1, 10 or 100 nMpladieno-
lide B for 4 h or left untreated. Unspliced mRNA was quantified using quantitative RT-PCR using primers specific for each intron. Each data
point represents the mean ± SD in triplicate.
SF3b as an antitumor target of pladienolide A. Yokoi et al.
4872 FEBS Journal 278 (2011) 4870–4880 ª2011 The Authors Journal compilation ª2011 FEBS
By contrast, the R1074H SF3B1 expressed ⁄endogenous
SF3B1-knockdown cells showed complete resistance to
cell growth suppression by pladienolide B. We further
assessed the inhibition of splicing by pladienolide B in
these transduced cells. Accumulation of unspliced pre-
mRNA of DNAJB1, CDKN1B and RIOK3 was not
observed in R1074H-expressing cells (Fig. 5B and Fig.
S2B). These results demonstrate that reverse expression
of SF3B1 (R1074H) transferred pladienolide resistance
to the cells.
The SF3B1 mutation impairs the binding of
pladienolide to SF3b
To further understand the mechanism of resistance in
pladienolide-resistant cells, we studied the interaction
of pladienolide with the SF3b complex containing
mutant SF3B1 using a radiolabeled probe. First, we
immunoprecipitated the SF3b complex from the
nuclear fraction of DLD1 and DLD1-R cells with
anti-SF3B1 serum. Western blot analysis showed that
the SF3b subunits SF3B1, SF3B2 and SF3B3 were
R-1DLD/1DLDR-rDiW/rDiW 3186
A
B
AAGGCTATTC
G
TAGAGCCACAGTCSF3B1
WiDr-R
cDNA
DLD1-R
cDNA
3260 3270 3280
Fig. 3. R1074H mutation detected in the gene for SF3B1. (A) Venn
diagram describing the number of mutations specific to each resis-
tant line from the mRNA-Seq differential analysis, in which mRNA
sequences were compared between parental and resistant pairs of
WiDr or DLD1 cells. (B) Illustration of the sequencing results. Con-
version of CGT into CAT in codon 1074, which results in the
replacement of an Arg with a His, was confirmed using capillary
sequencing.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
shLuc shSF3B1
(3989)
shSF3B1
(4145)
shLuc shSF3B1
(3989)
shSF3B1
(4145)
SF3B1 (Wt) SF3B1 (R1074H)
mRNA expression ratio
β-actin
HA
SF3B1
shLuc
shLuc
shSF3B1(3989)
shSF3B1(4145)
shSF3B1(3989)
shSF3B1(4145)
Wt R1074H
SF3B1 expression
Endogenous SF3B1
knockdown
CB
CMV
promoter HA-SF3B1 (Wt/R1074H)
RSV
promoter
shSF3B1 CMV
promoter
U6
promoter
Pol III
term
SF3B1 expression viral vector: pCLXIH-SF3B1(Wt or R1074H)
Endogenous SF3B1 knockdown viral vector: pLenti/shSF3B1(3989 or 4145)
A
(3989/4145)
Fig. 4. Establishment of WiDr cells expressing R1074H SF3B1. WiDr cells were transduced by the SF3B1 expression viral vector pCLXIH-
SF3B1 [wild-type (Wt) or R1074H] and the endogenous SF3B1 knockdown viral vector pLenti ⁄shSF3B1 (3989 or 4145). (A) Diagram of the
SF3B1 (Wt ⁄R1074H) expression viral vectors and the endogenous SF3B1 knockdown viral vectors. HA-SF3B1 indicates a HA-tagged SF3B1
cDNA. shSF3B1 indicates a shRNA template, which targets the 3¢-UTR of SF3B1. Targeting sequences of shSF3B1 (3989) and shSF3B1
(4145) correspond to nucleotides 3989–4009 and 4145–4165 in the gene for SF3B1, respectively. (B) Expression of SF3B1 was detected
using anti-HA and anti-SF3B1 sera. pLenti ⁄shLuc was used as a negative control for the knockdown viral vector. (C) Decreased expression
of endogenous SF3B1 was revealed using quantitative RT-PCR using primers designed for the 3¢-UTR region of the gene for SF3B1.
A. Yokoi et al. SF3b as an antitumor target of pladienolide
FEBS Journal 278 (2011) 4870–4880 ª2011 The Authors Journal compilation ª2011 FEBS 4873
coprecipitated equally from DLD1 and DLD1-R cells
(Fig. 6A). Next, we treated the immunoprecipitates
with a
3
H probe and evaluated the
3
H signal using
scintillation counting. The probe is a radiolabeled
derivative of pladienolide B, which was shown to com-
pete with a series of pladienolide for binding to the
SF3b complex [5]. Comparison of the radioactivity
between the DLD1 and DLD1-R cells showed that the
immunoprecipitate from the DLD1 cells retained 27
times the amount of
3
H probe retained in DLD1-R
cells (Fig. 6B). This demonstrated that the mutant
SF3B1 protein in DLD1-R reduced the ability of the
probe to bind to the SF3b complex.
Discussion
In the present study, we have shown that the targeting
of SF3b is the direct mechanism of the antitumor
activity of pladienolide. We first established pladieno-
lide-resistant lines (i.e. WiDr-R and DLD1-R) that
were resistant to the cell growth suppression and splic-
ing inhibition actions of pladienolide. The mRNA-Seq
differential analysis showed that the WiDr-R and
DLD1-R cells had a common mutation in the gene for
SF3B1 that resulted in the replacement of Arg1074
with a His. We next confirmed that reverse expression
of the mutant transferred the pladienolide resistance to
WiDr cells. Furthermore, immunoprecipitation analy-
sis with a radiolabeled probe showed that the binding
affinity of pladienolide to SF3b was impaired in
DLD1-R cells in which the R1074H SF3B1 was exclu-
sively expressed. These results clearly demonstrate that
pladienolide exerts its antitumor activity only by tar-
geting SF3b.
Although both WiDr-R and DLD1-R cells exhibited
resistance to pladienolide, the cell lines had different
characteristics. In WiDr-R cells, a high concentration
of pladienolide B suppressed cell proliferation by 30%,
whereas, in DLD1-R cells, no effect was noted at any
of the doses examined (Fig. 1B). The expression of
both wild-type and mutant-type of SF3B1 mRNA was
confirmed in WiDr-R cells, although only the mutant
0
20
40
60
80
100
120
0.01 0.1 1 10 100
Pladienolide B (nM)
Cell viability (% of control)
Wt
R1074H
B
CDKN1B
0
10
20
30
40
50
60
70
0100
Pladienolide B (nM)
Unspliced mRNA (fold change)
Wt
R1074H
0
2
4
6
8
10
12
14
16
18
0100
Pladienolide B (n
M
)
Wt
R1074H
0
1
2
3
4
5
6
7
8
9
10
0100
Pladienolide B (nM)
Wt
R1074H
RIOK3DNAJB1
A
Fig. 5. Reverse expression of SF3B1 (R1074H) transferred the pladienolide resistance to WiDr cells. (A) Antiproliferative activity of pladieno-
lide B against WiDr cells transduced by pCLXIH-SF3B1 [wild-type (Wt) or R1074H] and pLenti ⁄shSF3B1 (3989). Cell viability was assessed
after 72 h of treatment with pladienolide B using a colorimetric assay. Each data point represents the mean ± SD from three separate exper-
iments, each performed in triplicate. (B) Splicing inhibition by pladienolide B in WiDr cells transduced by pCLXIH-SF3B1 (Wt or R1074H) and
pLenti ⁄shSF3B1 (3989). Cells were treated with 100 nMof pladienolide B for 4 h. Unspliced DNAJB1 mRNA, CDKN1B mRNA and RIOK3
mRNA were quantified using quantitative RT-PCR using primers specific for each intron. Each data point represents the mean ± SD in
triplicate.
150
SF3B1
DLD1 DLD1-R
IP :
Cells :
150
150
SF3B1
SF3B2
SF3B3
*
**
**
*
0
100
200
300
400
500
600
700
DLD1 DLD1-R
Radio activity (Bq)
IP: SF3B1
AB
Fig. 6. The R1074H mutation in SF3B1 impairs the binding of pladi-
enolide to SF3b. (A) Immunoprecipitation of the SF3b components.
Anti-SF3B1 serum was used to immunoprecipitate (IP) DLD1 or
DLD1-R cells. Immunoblots were performed using anti-SF3B1, anti-
SF3B2 and anti-SF3B3 sera. Asterisks indicate proteins detected
nonspecifically. The molecular weight is indicated to the left. (B)
Radioactivity in the immunoprecipitated nuclear fraction. Samples
were immunoprecipitated with anti-SF3B1 serum and treated with
100 nM
3
H probe for 1 h. The
3
H signals of the immunoprecipitated
samples were measured using a liquid scintillation analyzer.
SF3b as an antitumor target of pladienolide A. Yokoi et al.
4874 FEBS Journal 278 (2011) 4870–4880 ª2011 The Authors Journal compilation ª2011 FEBS