Download Targeting RNA Polymerase I with an Oral Small Molecule CX

Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Cancer
Research
Therapeutics, Targets, and Chemical Biology
Targeting RNA Polymerase I with an Oral Small Molecule
CX-5461 Inhibits Ribosomal RNA Synthesis and Solid
Tumor Growth
Denis Drygin1, Amy Lin1, Josh Bliesath1, Caroline B. Ho1, Sean E. O’Brien1, Chris Proffitt1, Mayuko Omori1,
Mustapha Haddach1, Michael K. Schwaebe1, Adam Siddiqui-Jain1, Nicole Streiner1, Jaclyn E. Quin2,3, Elaine Sanij2,
Megan J. Bywater2,3, Ross D. Hannan2,3,4, David Ryckman1, Kenna Anderes1, and William G. Rice1
Abstract
Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA
polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus,
selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling
medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent
small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I–driven transcription
relative to Pol II–driven transcription, DNA replication, and protein translation. Molecular studies demonstrate
that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not
apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and
demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings
position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer
treatment. Cancer Res; 71(4); 1418–30. 2010 AACR.
Introduction
The rate of ribosome biogenesis controls cellular growth
and proliferation (reviewed in ref. 1). It, therefore, is tightly
regulated in mammalian cells and is tuned to respond to
extracellular stimuli such as nutrient availability and stress.
During tumorigenesis, the tightly regulated relationship
between extracellular signaling and ribosome biosynthesis
is disrupted, and cancer cells begin the excessive production of ribosomes necessary for the protein synthesis associated with unbridled cancer growth. rRNA is a major
component of the ribosome and, as such, carcinogenesis
requires an increase in its synthesis (reviewed in refs. 2–5).
Indeed, an increase in the synthesis of rRNA, which is
transcribed in the nucleolus by RNA polymerase (Pol) I,
correlates with an adverse prognosis in cancer (6). Moreover, enlarged nucleoli, reflective of accelerated rRNA
Authors' Affiliations: 1Cylene Pharmaceuticals, Inc., San Diego, California; 2Peter MacCallum Cancer Centre; 3Department of Biochemistry and
Molecular Biology, University of Melbourne; and 4Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria,
Australia
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Author: William G. Rice, Cylene Pharmaceuticals Inc.,
5820 Nancy Ridge Drive, Suite 200, San Diego CA 92121. Phone: 858875-5100; Fax: 858-875-5101. E-mail: [email protected]
doi: 10.1158/0008-5472.CAN-10-1728
2010 American Association for Cancer Research.
1418
synthesis, have long been recognized as a marker for
aggressive tumor cells (7, 8). A number of approved cancer
therapeutics reportedly act through inhibition of rRNA
synthesis, but none directly target the Pol I multiprotein
enzyme complex (4, 9).
The potential therapeutic benefit of selectively inhibiting
the Pol I target so fundamental to cancer cell survival
prompted the need for identification of small molecule
drugs that selectively inhibit rRNA synthesis. For this
purpose, we fashioned a cell-based quantitative reversetranscription PCR (qRT-PCR) assay that differentiates
between the effects of compounds on Pol I- and Pol II–
driven transcription. In this article, we describe the discovery and characterization of CX-5461, a potent and selective
inhibitor of Pol I–mediated rRNA synthesis in cancer cells
that does not inhibit DNA, mRNA or protein synthesis. We
reveal that CX-5461 induces autophagic cell death in cancer
cells but not normal cells and exhibits potent in vivo antitumor activity in murine xenograft models of human solid
tumors with a favorable safety profile. CX-5461 represents a
fundamentally new class of small molecule–targeted anticancer therapeutics.
Materials and Methods
Materials
CX-5461 and CX-5447 were synthesized by Cylene Pharmaceuticals as off-white solid materials (99.2%–99.5% pure) and
stored at room temperature as 10 mmol/L stock solutions in
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
50 mmol/L NaH2PO4 (pH 4.5). The compounds were diluted
directly in growth media prior to treatment.
Cell lines
hTERT-immortalized BJ-hTERT human fibroblasts were a
gift from Dr. William Hahn, Harvard Medical School (10).
Human inflammatory breast cancer cell lines SUM 149PT
and SUM 190PT were obtained from Asterand. Human
eosinophilic leukemia cell line EOL-1, human B cell precursor leukemia cell line SEM, and human acute monocytic
leukemia cell line THP-1 were obtained from DSMZ. Other
cell lines were purchased from American Type Culture
Collection. All cell lines were used within 5 to 10 passages
from their acquisition. The internal authentication has been
performed by monitoring growth rate and tracking the
changes in morphology.
qRT-PCR
qRT-PCR assays were performed as previously described
(11) with HCT-116 colorectal carcinoma cells and then confirmed with A375 and MIA PaCa-2 cells.
Cell-free Pol I transcription assay
A reaction mixture consisting of 30 ng/mL DNA template
corresponding to (160/þ379) region on rDNA and 3 mg/
mL nuclear extract isolated from HeLa S3 cells in a buffer
containing 10 mmol/L Tris-HCl, pH 8.0, 80 mmol/L KCl,
0.8% polyvinyl alcohol, 10 mg/mL a-amanitin was combined with different amounts of test compounds and incubated at ambient for 20 minutes. Transcription was
initiated by addition of rNTP mix (New England Biolabs)
to a final concentration of 1 mmol/L and was incubated for
1 hour at 30 C. Afterward, DNase I was added and the
reaction was further incubated for 2 hours at 37 C. DNase
digestion was terminated by the addition of EDTA to final
concentration of 10 mmol/L, followed immediately by 10minute incubation at 75 C, and then samples were transferred to 4 C. The levels of resultant transcript were analyzed by qRT-PCR on 7900HT Real Time PCR System
(Applied Biosystems).
Chromatin immunoprecipitation
Cells were treated with 2 mmol/L of CX-5461 for 1 hour and
chromatin immunoprecipitation (ChIP) assay was performed
as previously described (11, 12).
Electrophoretic mobility shift assay
32
P-labeled DNA probe corresponding to the rDNA
promoter was produced by PCR using prHu3 plasmid as
a template. The SL1 complex was isolated from HeLa
S3 cells nuclear extract as described previously (13). For
the competition studies, a mixture of 5 nmol/L of DNA
probe and 0.6 mg SL1 complex was incubated in binding
buffer for 15 minutes at ambient temperature with 0.4 to
12.5 mmol/L CX-5461 or CX-5447. The resulting complexes
were resolved using Novex TBE DNA retardation PAGE
(Invitrogen). The gels were dried and exposed to X-ray film
(Kodak).
www.aacrjournals.org
Gene expression analysis
For MIA PaCa-2 cells, gene array analyses were performed
at Expression Analysis using Illumina Whole Genome Human
-6 v2 beadchips. For A375 cells, the expression analysis was
performed using Human Cancer Pathway Finder qRT-PCR
array from SABiosciences.
Cell viability assay
Cells were plated on 96-well plates and treated the next day
with dose response of drugs for 96 hours. Cell viability
was determined using Alamar Blue and CyQUANT assays
(Invitrogen).
Detection and quantification of acidic vesicular
organelles with acridine orange staining
Cells were plated on 10-cm dishes and treated the next
day with indicated doses of CX-5461 for 24 or 48 hours. At
the end of treatment, cells were stained for 15 minutes with 1
mg/mL of acridine orange, trypsinized, washed twice with
ice-cold PBS, and analyzed on a BD LSR II flow cytometer
(BD Biosciences). The analysis was performed as previously
described (14).
Immunocytochemistry-based autophagy
detection assay
Immunofluorescent analysis was performed as previously
described (11) using 1:100 dilution of rabbit polyclonal antiLC3B-II antibody (Cell Signaling Technology).
Immunocytochemistry-based senescence
detection assay
The assay was performed using Senescence Detection Kit (Calbiochem) according to manufacturer's instructions.
In vivo efficacy in murine xenograft model
Animal experiments were performed with 5- to 6-weekold female athymic (NCr nu/nu fisol) mice of Balb/c origin
(Taconic Farms) in accordance with approved standard
operating protocols of Cylene Pharmaceuticals that were
approved by the Institutional Animal Care and Use Committee. Mice were inoculated with 5 106 in 100 mL of cell
suspension subcutaneously in the right flank. Tumor measurements were performed by caliper analysis, and tumor
volume was calculated using the formula (l w2)/2, where
w ¼ width and l ¼ length in mm of the tumor. Established
tumors (110–120 mm3) were randomized into vehicle
(50 mmol/L NaH2PO4, pH 4.5), gemcitabine, or CX-5461
treatment groups. Tumor growth inhibition (TGI) was
determined on the last day of study according to the
formula: TGI (%) ¼ [100 (VfD ViD)/ (VfV ViV) 100], where ViV is the initial mean tumor volume in vehicletreated group, VfV is the final mean tumor volume in
vehicle-treated group, ViD is the initial mean tumor volume
in drug-treated group, and VfD is the final mean tumor
volume in drug-treated group.
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1419
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
Results
Discovery of CX-5461
To screen for inhibitors of rRNA synthesis, we developed a
cell-based screening assay capable of identifying agents that
selectively inhibit Pol I transcription relative to Pol II transcription. Two short-lived RNA transcripts (half-lives 20–30
minutes), one produced by Pol I and another by Pol II, were
quantitated by qRT-PCR as a measure of drug-related effects
on transcription. The 45S pre-rRNA served as the Pol I
transcript and the mRNA for the protooncogene c-myc served
as the comparator Pol II transcript. Both Pol I and Pol II
transcription are known to be affected by general cellular
stress. To minimize the potential effects of such stress, cells
were exposed to test agents for only a short period of time (2
hours). This is sufficient time for these transcripts to be
reduced by greater than 90% if a drug affects their synthesis.
Among numerous molecules screened, CX-5461 (Fig. 1A)
was found to selectively inhibit rRNA synthesis (Pol I IC50 ¼ 142
nmol/L; Pol II IC50 > 25 mmol/L; selectivity 200-fold) in the
HCT-116 cells (Fig. 1B). Selective inhibition of rRNA synthesis
by CX-5461 was confirmed in two other human solid tumor cell
lines; melanoma A375 (Pol I IC50 ¼ 113 nmol/L; Pol II IC50 > 25
mmol/L) and pancreatic carcinoma MIA PaCa-2 (Pol I IC50 ¼ 54
nmol/L; Pol II IC50 25 mmol/L; Fig. 1C).
Characterization of CX-5461
To determine if CX-5461 directly targets the Pol I machinery
and to identify which specific step in Pol I transcription it may
affect, we performed cell-free transcription "order of addition"
studies (Fig. 1D). CX-5461 was preincubated with either
nuclear extract (NE) or with DNA template (Template) prior
to the preinitiation complex (PIC) formation, after the PIC
formation but prior to initiation of transcription (NE/Template), or after the initiation of the transcription (NE/Template/NTP). Analysis of the resulting rRNA transcripts by qRTPCR (Fig. 1D) revealed that addition of 80 nmol/L CX-5461
prior to PIC formation resulted in considerable inhibition of
Pol I transcription, whereas addition after PIC formation only
minimally affected Pol I transcription. These data indicate CX5461 directly targets the Pol I machinery and inhibits Pol I
transcription at the initiation stage.
CX-5461 inhibits Pol I via disruption of the SL1-rDNA
complex
ChIP analysis was employed to investigate the effects of CX5461 on the association of various components of the Pol I
multiprotein complex with the rDNA promoter. Treatment of
HCT-116, A375, or MIA PaCa-2 with 2 mmol/L CX-5461
resulted in 40% to 60% reduction of the Pol I enzyme association with the rDNA promoter (Fig. 2A). Further, CX-5461
significantly depleted the binding of Pol I transcription factors
(TF) to the rDNA promoter in HCT-116 cells, with the TBP and
TAFI110 subunits of SL1 being most overtly affected (Fig. 2B).
As SL1 is required for stabilization of UBF on and recruitment
of Pol I to the rDNA promoter, the effect of CX-5461 on the
binding of UBF and Pol I to rDNA is most likely secondary to
the compound's effect on SL1 (15).
1420
Cancer Res; 71(4) February 15, 2011
Because TBP is a member of both Pol I and Pol II transcription machineries (as a unit of the TFIID transcription
factor), we compared the effects of CX-5461 on binding of TBP
with the Pol I promoter on rDNA relative to its binding to the
promoter of 2 genes transcribed by Pol II (p21 and histone
H2B). Treatment with CX-5461 had no effect on the binding of
TBP to the Pol II promoter regions in HCT-116 cells, but did
inhibit binding of TBP to the Pol I promoters (Fig. 2C). To test
if CX-5461 could directly compete with SL1 for the rDNA
promoter, we performed an electrophoretic mobility shift
assay (EMSA) measuring the interaction of purified human
SL1 with a radiolabeled, double-stranded DNA fragment corresponding to the promoter region of rDNA. As a negative
control, we used a close analogue of CX-5461, CX-5447, that is
inactive against Pol I transcription (IC50 > 25 mmol/L).
Although CX-5447 had no detectable effect on the stability
of the SL1/rDNA complex, CX-5461 clearly disrupted the SL1/
rDNA complex (Fig. 2D). Such dissociation was likely due to
disruption of the protein–rDNA interaction rather than dissociation of SL1 itself, as we detected no decrease in protein–
protein interactions within SL1 after treatment with CX-5461
(data not shown).
Biological characterization of CX-5461
To ensure that CX-5461 selectively inhibits rRNA transcription but not DNA or protein synthesis, HCT-116, A375, and
MIA PaCa-2 cells were preincubated with 10 mmol/L CX-5461
for 60 minutes and pulsed for an additional 120 minutes with
modified precursors BrdU or 35S-methionine. Mitoxantrone
and cycloheximide served as positive controls for inhibition
of DNA and protein synthesis, respectively. At this concentration, which is approximately 100-times higher than its IC50
for the inhibition of Pol I transcription, CX-5461 had only
modest effects on global DNA or protein synthesis (Fig. 3A).
In dose–response studies, the IC50 for inhibition of DNA
synthesis in A375 and MIA PaCa-2 cell lines ranged from
16.8 to 27.9 mmol/L, whereas a high-test dose of 30 mmol/L
CX-5461 had no significant effect on proteins synthesis (Supplementary Fig. S1). Thus, CX-5461 possesses 250- to 300-fold
selectivity for inhibition of rRNA transcription versus DNA
replication and protein translation.
Using Gene Expression Arrays (Illumina or qRT-PCR based)
as a tool to probe for any potential effect of CX-5461 on Pol II
transcription, we observed that 1-hour exposure of MIA PaCa2 (Fig. 3B) or A375 (Fig. 3C) cells to 300 nmol/L CX-5461 (that
resulted in 63% and 55% reductions in pre-rRNA, respectively),
an equal number of Pol II–transcribed genes were significantly
upregulated as were downregulated. These changes in Pol II
transcription were most likely secondary to the inhibition of
Pol I by CX-5461, as an inhibition of Pol II transcription results
in a distribution significantly skewed to the negative (16). To
further illustrate selectivity of CX-5461 for Pol I versus Pol II
transcription, we compared it with another drug (actinomycin
D) known to inhibit Pol I transcription. Treatment of MIA
PaCa-2 cells with either 1 mmol/L CX-5461 or 1 mmol/L
actinomycin D causes rapid inhibition of Pol I transcription,
with an observed half-life of 21 minutes for 45S pre-rRNA in
both cases (Fig. 3D). However, actinomycin D inhibited Pol II
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
A
B
N
N
c-myc mRNA
N
S
pre-rRNA
150
N
N
N
HCT-116
O
Rel RNA levels (%)
O
N
100
50
0
–9
–8
–7
–6
log ([CX-5461])
C
A375
pre-rRNA
Rel RNA levels (%)
Rel RNA levels (%)
c-myc mRNA
150
β-Actin mRNA
100
50
0
β-Actin mRNA
100
50
0
–10
D
–4
MIA PaCa-2
pre-rRNA
c-myc mRNA
150
–5
–8
–6
log ([CX-5461])
–4
–10
–8
–6
log ([CX-5461])
–4
20’
5’
45’
+ Template
+ NTP
Analysis
5’
45’
20’
(II) Template + CX-5461
+ NE
+ NTP
Analysis
5’
45’
20’
+ NTP
Analysis
(III) NE + Template
+ CX-5461
(I) NE + CX-5461
(IV) NE + Template
Inhibition of Transcription (%)
50
20’
+ U/G/CTPs 20’ + CX-5461 5’
Pre-PIC formation
+ NTP 45’ Analysis
Post-PIC formation
40
30
20
10
0
I
II
III
IV
NE
TEMPLATE
NE
TEMPLATE
NE
TEMPLATE
NTP
Figure 1. CX-5461 selectively inhibits rRNA synthesis. A, structure of CX-5461. B, inhibition of Pol I (pre-rRNA) versus Pol II (c-myc mRNA) transcription
in HCT-116 cells by CX-5461. C, qRT-PCR analysis of rRNA, c-myc mRNA, and b-actin mRNA transcription after 2-hour treatment with CX-5461
in A375 and MIA PaCa-2 cells. D, order of addition study setup and results.
www.aacrjournals.org
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1421
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
B
1.5
UTC
CX-5461
1.0
0.5
0.0
UTC
CX-5461
1.0
0.5
BF
63
U
TA
Fi
11
0
TB
P
a2
Pa
C
M
IA
H
C
T11
6
A3
75
0.0
TA
Fi
Relative Pol I ChIP recovery
1.5
Relative TF ChIP recovery
A
C
Relative TBP ChIP recovery
2.0
UTC
CX-5461
1.5
1.0
0.5
p2
rR
H
N
A
2B
1
0.0
CX-5461
0.4 0.8 1.6 3.2
150
CX-5447
6.3 12.5 0.4 0.8 1.6 3.2 6.3 12.5 µmol/L
SL1-rDNA
rDNA
% Untreated
DMSO
D
CX-5461
CX-5447
100
50
0.
4
0.
8
1.
6
3.
1
6.
3
12
.5
0.
4
0.
8
1.
6
3.
1
6.
3
12
.5
0
Drug (µmol/L)
Figure 2. CX-5461 selectively competes SL1 from the Pol I promoter on rDNA. A, ChIP analysis of Pol I associations with Pol I promoter in HCT-116,
A375, and MIA PaCa-2 cells. B, ChIP analysis of Pol I TF SL1 (TBP, TAFI110, TAFI63) and UBF associations with Pol I promoter in HCT-116 cells. C, ChIP
analysis of TBP associations with Pol I, histone H2B, and p21 promoters in HCT-116 cells. D, EMSA of SL1-rDNA complex in presence of increasing
concentrations of CX-5461 and CX-5447. UTC, untreated control.
transcription as well, as judged by the depletion of c-myc
mRNA (half-life ¼ 35 minutes), whereas CX-5461 had no major
effect on Pol II transcription even after 24-hour treatment
(Fig. 3D).
1422
Cancer Res; 71(4) February 15, 2011
In vitro antiproliferative activity of CX-5461
To evaluate the range of antiproliferative activity of CX5461, we measured the antiproliferative EC50 across a panel of
50 human cancer cell lines and 5 nontransformed cell lines.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
UTC
UTC
150
10 µmol/L CX-5461
1 µmol/L Mitoxantrone
100
50
1 µmol/L Cycloheximide
100
50
20
10
1.0
Pa
C
C
1.5
M
IA
H
M
IA
% of total genes
% of total genes
40
30
0.5
a2
T11
6
a2
Pa
C
A3
T11
6
C
H
C
40
0
−2.0 −1.5 −1.0 −0.5 0.0
75
0
75
0
B
10 µmol/L CX-5461
A3
DNA synthesis (%)
150
Protein synthesis (%)
A
30
20
10
0
−2.0 −1.5 −1.0 −0.5
2.0
log2 fold change
0.0
0.5
1.0
1.5
2.0
log2 fold change
D
150
1 µmol/L CX-5461
1 µmol/L act D
80
cMyc mRNA Levels (%)
pre-rRNA levels (%)
100
60
40
20
0
1 µmol/L CX-5461
1 µmol/L act D
100
50
0
0
500
1,000
1,500
Time (min)
2,000
0
500
1,000
1,500
Time (min)
2,000
Figure 3. CX-5461 inhibits Pol I transcription without having an effect on DNA replication, protein translation, and general cellular transcription. A, effect
of CX-5461 on total DNA and protein synthesis in HCT-116, A375 and MIA PaCa-2 cells. Histogram showing the proportion of genes that were up- or
downregulated upon treatment of (B) MIA PaCa-2 or (C) A375 cells with 300 nmol/L of CX-5461 for 1 hour. Each column represents a bin of 0.25 units
(log2 change in gene expression) and its height is proportional to the number of genes showing such a change in detected mRNA levels. D, comparative
analysis of the effects of CX-5461 and actinomycin D (act D) on Pol I– and Pol II–driven transcription.
The EC50 values ranged from 3 nmol/L against the EOL-1
eosinophilic leukemia cell line to 5,500 nmol/L against the
LNCaP prostate carcinoma cell line (Fig. 4A). The median EC50
across all tested cell lines was 147 nmol/L, yet all normal cell
lines had EC50 values of approximately 5,000 nmol/L. Evaluation of the antiproliferative dose response (Fig. 4B, left) for
HCT-116, A375, and MIA PaCa-2 cell lines yielded EC50 values
of 167, 58, and 74 nmol/L, respectively. In contrast, an EC50 of
www.aacrjournals.org
approximately 5,000 nmol/L was observed for the BJ-hTert
normal cell line (nontransformed hTERT-immortalized
human foreskin fibroblasts). The lesser sensitivity of the
normal cells was not due to a reduced uptake of CX-5461,
as the IC50 for rRNA synthesis for the BJ-hTert normal cell line
of 74 nmol/L correlated well with the IC50 values of the solid
tumor cell lines (IC50 ¼ 142, 113, and 54 nmol/L, respectively;
see Fig. 4B, right). These findings illustrate that CX-5461 can
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1423
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
A
10,000
Cancer
Normal
EC50 (nmol/L)
1000
Median = 147 nmol/L
100
10
EO
L1
M SR
V4
1
SE 1
M
N
C A
I-H 7
4
N TH 60
C P
I-H 12 1
9
A3 9
7
Ju 5
r
R Ra kat
PM m
o
N I-8 s
M CI- 226
IA H
Pa 520
SK Ca-O 2
M
DA H V-3
-M L6
B- 0
2
C BT- 31
O 4
LO 74
-2
05
H K5
s 62
6
ZR 05.
-7 T
51
R
M
DA SK aji
-M Br
B- 3
4
H D 53
L a
SK 60/ ndi
-M MX
E 2
H L-2
C 4
T
M N -11
DA K 6
-M 92
N B- mi
C 4
I-H 68
21
U 70
2O
BT S
SU M -20
M CF
19 7
Bx 0P
PC T
SU
-3
M HT14 29
9P
SK P T
-M C-3
H ES
s
U 5 -1
M AC 78
DA C .T
-M -81
M
B- 2
DA
36
-M
T 1
C B-1 47
C 7 D
C D-1 5-V
C 0 II
D 58
-1 S
09 k
4S
A k
Sa 54
9
C PA osC N 2
D C
-1 BJ 068 1
C -h S
C T k
D E
-1 R
09 T
6
LN Sk
C
aP
1
B
HCT-116 (IC50 = 162 nmol/L)
A375 (IC50 = 58 nmol/L)
MIA PaCa-2 (IC50 = 74 nmol/L)
BJ-hTERT (IC50 = 5174 nmol/L)
100
50
0
−10
−9
−8
−7
−6
HCT-116 (IC50 = 142 nmol/L)
A375 (IC50 = 113 nmol/L)
MIA PaCa-2 (IC50 = 54 nmol/L)
BJ-hTERT (IC50 = 74 nmol/L)
150
% Pol l transcription
% Cell viability
150
−5
−4
100
50
0
−10
−8
−6
C
D
0.1
0.3
48 h
1.0
0.1
10
0.3
(µmol/L) CX-5461
1.0
10
(nmol/L) actinomycin D
p53
Tubulin
P value
0.5629
10,000
CX-5461 IC50 (nmol/L)
24 h
−4
log [CX-5461]
log [CX-5461]
1,000
100
10
M
W
T
ut
1
p53 Status
Figure 4. CX-5461 exhibits broad antiproliferative potency in a panel of cancer cell lines in p53-independent manner, but has minimal effect on viability
of nontransformed human cells. A, panel of cancer and normal cell lines were treated with various doses of CX-5461 for 96 hours and the resulting
effects on cell viability were measured with CyQUANT assay. B, effect of CX-5461 on cell viability and Pol I transcription of HCT-116, A375, MIA PaCa-2, and
BJ-hTERT cells. C, effect of CX-5461 on p53 stabilization in A375 cells. D, CX-5461 exhibit similar activity against p53 wt and p53 mutant solid cancer cell lines.
1424
Cancer Res; 71(4) February 15, 2011
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
equivalently inhibit Pol I transcription in cancer and normal
cells, but the normal cells can tolerate reductions in rRNA
synthesis without induction of cell death whereas the cancer
cells cannot.
Inhibition of Pol I transcription has been previously
demonstrated to cause nucleolar stress that leads to stabilization of p53 and induction of p53-dependent apoptosis
(17). We therefore wanted to determine if CX-5461 promotes
stabilization of p53. For this purpose, we performed a dose–
response analysis of CX-5461 and measured levels of p53 at
24 and 48 hours in wild-type (wt) p53 A375 cells. As shown in
Figure 4C, whereas CX-5461 slightly increased p53 levels, it
did so at concentrations 3- to 10-fold higher than its IC50 for
Pol I transcription. Actinomycin D, used as a positive control, caused a more robust increase in p53 levels. In addition,
we analyzed the correlation between the sensitivity of cell
lines to CX-5461 and their p53 genetic status. Among the 44
cell lines with known p53 status, 18 had wt p53 and 26
carried some form of mutation in the p53 gene (18). The
analysis revealed that across all tested cell lines CX-5461
exhibits similar sensitivity in wt p53 cell lines (median IC50 ¼
144 nmol/L) as it does in cell lines with mutant p53 (median
IC50 ¼ 138 nmol/L), P ¼ 0.56 (Fig. 4D). Interestingly, the 3
cell lines most sensitive to CX-5461 were derived from
hematologic malignancies that had wt p53. When we then
further analyzed data for correlations of p53 status with
sensitivity to CX-5461 among hematologic cancers, we did
observe a significant difference (P ¼ 0.003) between the
activity of CX-5461 in cell lines with wt p53 (3 cell lines,
median IC50 ¼ 5 nmol/L) and those with mutant p53 (8 cell
lines, median IC50 ¼ 94 nmol/L). Although the sample size is
too limited to make strong conclusions, it does hint at an
intriguing possibility that nucleolar stress signaling in hematologic cancers is more dependent on p53 function and is
worthy of future investigation.
CX-5461 induces autophagy and senescence in solid
tumor cancer cells
Next, we investigated the mode of cell death promoted by
CX-5461 in the A375 and MIA PaCa-2 cells. Previously, inhibition of rRNA synthesis using "semi-selective" drugs, such as
actinomycin D, was shown to induce apoptosis (19). Not
surprisingly, we found that actinomycin D induced apoptosis,
as judged by Western blot analysis of PARP cleavage and
caspases-3/7/9 cleavage (Fig. 5A), as well as caspase activation
analysis and Terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) assay (Supplementary Fig. S2). However,
no changes in these apoptotic markers were observed for CX5461, with the exception of very modest cleavage of PARP after
48-hour treatment with 1 mmol/L of CX-5461 in MIA PaCa-2
(Fig. 5A). These data indicated that apoptosis is not the
primary pathway through which CX-5461 affected cellular
viability. In addition, no evidence of necrotic cell death was
detected, as measured by lactate dehydrogenase (LDH) release
as late as 48 hours after the initiation of treatment (data not
shown). These data led us to consider the possibility that CX5461 might be inducing autophagy to kill these solid tumor
cell lines.
www.aacrjournals.org
Using immunofluorescence cytometry to monitor the production of LC3B-II, a known marker of autophagy (20), we
observed that a 24-hour treatment with 300 nmol/L CX-5461
caused a significant increase from 15% 3% to 67% 2% in
LC3B-II staining (P ¼ 0.0041) in A375 and from 19% 2% to
62% 3% in LC3B-II staining (P ¼ 0.002) in MIA PaCa-2 cells
(Fig. 5B). In addition, CX-5461 promoted a concentrationdependent increase in acidic vesicular organelles (AVO)
including autophagolysosomes, another marker of autophagic
response (refs. 14, 21; Fig. 5C, see Supplementary Materials
and Methods). To exclude the possibility that induction of
autophagy by CX-5461 resulted from a decrease in the protein
translation rate, we performed a time-course analyses to
monitor the effects of CX-5461 on protein synthesis in A375
and MIA PaCa-2 cells (Supplementary Fig. S3). Although 24hour treatment of A375 or MIA PaCa-2 cells with CX-5461
dramatically reduced Pol I transcription (Fig. 3D; data not
shown), it had only a minor effect on overall protein synthesis
(13%–19% reduction at 300 nmol/L and 24%–27% reduction at
1 mmol/L). Several reports that have investigated the effects of
overall protein synthesis inhibition on the viability of cancer
cells demonstrated that such a reduction (i.e., <30%) should
not have a significant effect on cell viability (22). As autophagic
cells can themselves have reduced protein synthesis rate (23),
the observed effect of CX-5461 on protein synthesis is likely
secondary to the induction of autophagy and not vice versa.
Several recent reports link autophagy to the induction of
cellular senescence (24). The analysis of senescence-associated marker b-galactosidase demonstrated that, in addition
to autophagy, CX-5461 is able to induce senescence in both
cell lines. Treatment with 300 nmol/L CX-5461 for 24 hours
caused an increase in b-galactosidase staining from 6% 1%
to 79% 1% (P < 0.0001) in A375 cells and from 14% 2% to
55% 3% (P < 0.0001) in MIA PaCa-2 cells (Fig. 6). Together,
the mechanistic data demonstrate that treatment of solid
tumor cell lines with CX-5461 induces cellular senescence and
autophagy through selective inhibition of rRNA synthesis.
In vivo antitumor activity of CX-5461
The antitumor activity of CX-5461 was evaluated in two
murine xenograft models of human cancers, pancreatic carcinoma (MIA PaCa-2) and melanoma (A375). In these xenograft models, CX-5461 was administered orally (50 mg/kg)
either once daily or every 3 days. Untreated animals received
vehicle orally on the equivalent schedule, whereas the positive
control gemcitabine was administered intraperitoneally once
every 3 days at 120 mg/kg. CX-5461 demonstrated significant
MIA PaCa-2 TGI with TGI equal to 69% on day 31 (Fig. 7A),
comparable to that of gemcitabine (63% TGI). Likewise, CX5461 demonstrated significant A375 TGI (Fig. 7B) with TGI
equal to 79% on day 32. Unpaired t test revealed statistically
significant differences between the vehicle-treated and CX5461–treated groups throughout both studies. CX-5461 was
well tolerated at all tested schedules as judged by the absence
of significant changes in animal body weights. These data
demonstrate that a selective inhibitor of Pol I transcription
can produce in vivo antitumor responses against solid tumors,
with a favorable therapeutic window.
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1425
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
A
A375
24 h
0.1 0.3 1.0
MIA PaCa-2
48 h
0.1 0.3 1.0
10
24 h
0.1 0.3 1.0
10
10
48 h
0.1 0.3 1.0
10
(µmol/L) CX-5461
(nmol/L) actinomycin D
PARP
Cleaved PARP (D214)
Caspase-3
Cleaved caspase-3 (D175)
Caspase-7
Cleaved caspase-7 (D198)
Caspase-9
Cleaved caspase-9 (D330)
Caspase-8
Cleaved caspase-9 (D391)
B
MIA PaCa-2
300 nmol/L CX-5461
UTC
A375
C
A375
24 h
48 h
4
**
4
**
**
**
2
*
0
Mean red:green
fluorescence (fold UTC)
Mean red:green
fluorescence (fold UTC)
6
MIAPaCa-2
24 h
48 h
3
***
***
*
**
2
*
1
/L
/L
ol
ol
1,
00
0n
m
m
0n
54
61
30
X54
61
10
C
C
X-
0n
m
ol
/L
TC
m
U
54
61
XC
C
X54
61
1,
00
30
61
54
XC
0n
0n
m
m
0n
10
ol
/L
ol
/L
ol
TC
U
61
C
X54
/L
0
Figure 5. CX-5461 induces autophagy but not apoptosis in A375 and MIA Paca-2 cancer cells. A, A375 and MIA PaCa-2 cells were treated with
various doses of CX-5461 for 24 hours and the induction of apoptosis was measured by monitoring activation of caspases-3/7/8/9 and PARP cleavage.
Actinomycin D (act D) was used as a positive control. B, A375 and MIA PaCa-2 cells were treated with 300 nmol/L of CX-5461 for 24 hours and the induction of
autophagy was measured by detection of LC3B-II using fluorescent microscopy. C, A375 and MIA PaCa-2 cells were treated with a dose response of
CX-5461 for 24 or 48 hours and the induction of autophagy was measured by acridine orange staining followed by flow cytometry. Statistically significant
differences from untreated control (UTC) are marked by asterisks (unpaired two-tailed t-test; *, P < 0.05; **, P < 0.01, ***, P < 0.001).
1426
Cancer Res; 71(4) February 15, 2011
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
MIA PaCa-2
Figure 6. CX-5461 induces
senescence in A375 and MIA
Paca-2 cancer cells. A375 and
MIA PaCa-2 cells were treated
with 300 nmol/L of CX-5461 for 24
hours and the induction of
senescence was measured by
b-galactosidase staining.
300 nmol/L CX-5461
UTC
A375
Discussion
Deregulated rRNA synthesis plays a fundamental role in
tumorigenesis (reviewed in refs. 1–5), and Pol I is the key
regulatory enzyme required for production of rRNA (reviewed
in refs. 25–27). Herein, we present the results from a discovery
program that identified CX-5461, a small molecule that selectively inhibits Pol I–driven transcription of rRNA but does not
inhibit Pol II–driven mRNA synthesis or DNA or protein
synthesis. CX-5461 must be viewed globally as targeting a
specific transcriptional event. A host of currently marketed
drugs target transcription, including tamoxifen, cyclosporins,
salicylates, and others (28). In the case of CX-5461, it disrupts
the binding of the SL1 transcription factor to rDNA promoter,
which inhibits initiation of rRNA synthesis by the Pol I multiprotein complex. The inhibition of rRNA synthesis leads to
induction of senescence and autophagy in a p53-independent
manner in solid tumor cell lines. This response is not driven by
reductions in ribosomes or protein synthesis, as the cancer
cell induces the death pathway signaling long before any
reduction in ribosomes or protein content can occur (29).
SL1, a protein complex containing TATA-binding protein–
associated factors, is responsible for Pol I promoter specificity
(27, 30, 31). SL1 performs important tasks in transcription
complex assembly, mediating specific interactions between
the rDNA promoter region and the Pol I enzyme complex,
thereby recruiting Pol I, together with a collection of Pol I–
associated factors, to rDNA. Our data indicate that disruption
www.aacrjournals.org
of the SL1/rDNA complex by CX-5461 results from the interference between SL1 and rDNA and not through dissociation
of protein–protein interactions, as was shown for several
known tumor suppressors (p53, Rb, PTEN; refs. 32–34).
Numerous reports have linked the inhibition of rRNA
synthesis to induction of apoptosis. Actinomycin D, a relatively selective inhibitor of rRNA synthesis, inhibits Pol I
transcription during the elongation step (35) and induces
apoptosis in cancer cell lines (36). Inhibition of Pol I transcription at the elongation step leads to the formation of
truncated pre-rRNA, which can be interpreted by the cell as
breaks in rDNA. The cell attempts to repair such breaks and
ultimately triggers the apoptotic response (37). In contrast,
CX-5461 inhibits Pol I transcription prior to/during the PIC
formation step and induces autophagy in solid tumor cell
lines. When Pol I transcription is inhibited at the initiation
step, cells may interpret this as decreased nutrient availability.
The process of autophagy is known to be used by cancer cells
to survive times of limited nutrient availability, but when
driven to the extreme it causes cell death (38). Cancer cells are
continuously subjected to autophagy inducing stress (e.g.,
unfolded protein response; reviewed in ref. 39), and this
may prime them to become more susceptible to certain types
of metabolic stress such as inhibition of rRNA synthesis. This
may be one of the reasons why rapamycin that targets mTOR,
a protein kinase that serves as a sensor for nutrient availability
and in turn controls both rRNA as well as protein synthesis
(40), can also induce autophagy (reviewed in ref. 41). Likewise,
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1427
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
A
Vehicle
CX-5461 50 mg/kg q.d. PO
Gemcitabine 120 mg/kg q3d IP
1,200
Tumor volume (mm3)
Body weight (g)
30
25
20
800
15
0
10
20
Days
30
40
400
0
0
10
20
30
40
Days
B
Vehicle
CX-5461 50 mg/kg q3d PO
Gemcitabine 120 mg/kg q3d IP
1,500
Tumor volume (mm3)
Body weight (g)
30
1,000
Figure 7. CX-5461 demonstrates
in vivo anticancer activity. MIA
PaCa-2 human pancreatic cancer
(A) and A375 human melanoma (B)
xenograft models in which mice
were treated with vehicle, CX5461, or gemcitabine. The
resulting changes in tumor
volumes and body weights are
demonstrated. Statistical
significance in tumor volume
between vehicle-treated and CX5461–treated groups is marked by
asterisks (*, P < 0.05; **, P < 0.01).
25
20
15
0
10
20
30
40
Days
500
0
0
1428
10
Cancer Res; 71(4) February 15, 2011
20
Days
30
40
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Selective Inhibitor of RNA Pol I Complex Exhibits Antitumor Activity
this might illustrate how CX-5461 takes advantage of the
unique qualities of cancer cells and elicits potent and selective
antitumor activity, whereas having minimal effect on nontransformed cells.
CX-5461 exhibited a broad-spectrum antiproliferative activity of cancer cells in vitro, whereas having a lesser effect on the
viability of nontransformed cells, indicating that certain cancer cells are significantly more susceptible to inhibition of
rRNA synthesis than nontransformed cells. Further, we analyzed if sensitivity of CX-5461 correlates with the p53 mutational status of cells, as inhibition of Pol I was previously
reported to cause nucleolar stress that leads to stabilization of
p53 and induction of p53-dependent apoptosis (17). Across all
cell lines, CX-5461 exhibited similar sensitivity in wt p53 and
mutant p53 cell lines. However, wt p53 cell lines derived from
hematologic malignancies appeared highly sensitive to CX5461 and this apparent trend is the focus of additional
investigations.
Herein, we present the results of a discovery and development program that identified CX-5461, a small molecule that
selectively inhibits Pol I transcription of rRNA. CX-5461 does
not inhibit Pol II transcription of mRNA or inhibit the synthesis of DNA or proteins. CX-5461 targets the SL1 transcription
factor of the Pol I complex and induces autophagy and
senescence among solid tumor cell lines and selectively kills
cancer cells relative to normal cells. Studies with human solid
tumors grown in murine xenograft models revealed that CX5461 can be orally administered with favorable pharmacokinetics and an antitumor efficacy without changes in body
weight or overt toxicity. In addition, our data demonstrate
that the anticancer activity of CX-5461 against solid tumors
does not depend on the p53 pathway that is frequently
mutated in many types of cancer. Thus, CX-5461 represents
a first-in-class oral small molecule therapeutic agent that
selectively targets Pol I transcription and induces autophagy
in solid tumors.
Disclosure of Potential Conflicts of Interest
D. Drygin, J. Bliesath, C.B. Ho, S. O’Brien, C. Proffitt, M. Omori, M. Haddach,
M. Schwaebe, A. Siddiqui-Jain, N. Streiner, D. Ryckman, K. Anderes and W.G.
Rice own stock options in Cylene Pharmaceuticals. Cylene Pharmaceuticals has
a pending patent application covering composition of matter for CX-5461. M.
Haddach and M. Schwaebe are named among the inventors. The other authors
declared no potential conflicts of interest.
Acknowledgments
We thank Mr. Jerome Michaux for his help in synthesizing CX-5461,
Dr. William Hahn from Harvard Medical School for his kind gift of BJ-hTERT
cells, and Dr. Lucio Comai from Keck Medical School for his kind gift of
antibodies to SL1 subunits.
Grant Support
This work was supported in part by Cylene Pharmaceuticals Inc. and in part
by grants to R.D. Hannan and E. Sanij from the National Health and Medical
Research Council (NHMRC) of Australia. R.D. Hannan was supported by a
research fellowship from the NHMRC. M.J. Bywater was supported by an
NHMRC postgraduate scholarship. J.E. Quin was supported by an Australian
postgraduate award.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received May 14, 2010; revised November 11, 2010; accepted November 15,
2010; published OnlineFirst December 15, 2010.
References
1.
Ruggero D, Pandolfi PP. Does the ribosome translate cancer?Nat Rev
Cancer 2003;3:179–92.
2. White RJ. RNA polymerases I and III, growth control and cancer. Nat
Rev Mol Cell Biol 2005;6:69–78.
3. White RJ. RNA polymerases I and III, non-coding RNAs and cancer.
Trends Genet 2008;24:622–9.
4. Drygin D, Rice WG, Grummt I. The RNA polymerase I transcription
machinery: an emerging target for the treatment of cancer. Annu Rev
Pharmacol Toxicol 2010;50:131–56.
5. Silvera D, Formenti SC, Schneider RJ. Translational control in cancer.
Nat Rev Cancer 2010;10:254–66.
6. Williamson D, Lu YJ, Fang C, Pritchard-Jones K, Shipley J. Nascent
pre-rRNA overexpression correlates with an adverse prognosis in
alveolar rhabdomyosarcoma. Genes Chromosomes Cancer 2006;
45:839–45.
7. Derenzini M, Trere D, Pession A, Govoni M, Sirri V, Chieco P. Nucleolar
size indicates the rapidity of cell proliferation in cancer tissues. J
Pathol 2000;191:181–6.
8. Maggi LB Jr., Weber JD. Nucleolar adaptation in human cancer.
Cancer Invest 2005;23:599–608.
9. Burger K, Muhl B, Harasim T, et al. Chemotherapeutic drugs inhibit
ribosome biogenesis at various levels. J Biol Chem 2010;285:12416–25.
10. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW,
Weinberg RA. Creation of human tumour cells with defined genetic
elements. Nature 1999;400:464–8.
11. Drygin D, Siddiqui-Jain A, O’Brien S, et al. Anticancer activity of CX3543: a direct inhibitor of rRNA biogenesis. Cancer Res 2009;
69:7653–61.
www.aacrjournals.org
12. Lin CY, Navarro S, Reddy S, Comai L. CK2-mediated stimulation of
Pol I transcription by stabilization of UBF-SL1 interaction. Nucleic
Acids Res 2006;34:4752–66.
13. Comai L, Tanese N, Tjian R. The TATA-binding protein and associated
factors are integral components of the RNA polymerase I transcription
factor, SL1. Cell 1992;68:965–76.
14. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells
to radiation involves autophagy and formation of acidic vesicles.
Cancer Res 2001;61:439–44.
15. Friedrich JK, Panov KI, Cabart P, Russell J, Zomerdijk JC. TBP-TAF
complex SL1 directs RNA polymerase I pre-initiation complex formation and stabilizes upstream binding factor at the rDNA promoter. J
Biol Chem 2005;280:29551–8.
16. Vispe S, DeVries L, Creancier L, et al. Triptolide is an inhibitor of RNA
polymerase I and II-dependent transcription leading predominantly to
down-regulation of short-lived mRNA. Mol Cancer Ther 2009;8:2780–
90.
17. Kalita K, Makonchuk D, Gomes C, Zheng JJ, Hetman M. Inhibition of
nucleolar transcription as a trigger for neuronal apoptosis. J Neurochem 2008;105:2286–99.
18. Catalogue of Somatic Mutations in Cancer. Wellcome Trust Genome
Campus, Hinxton, Cambridge, UK; 2010.
19. Martin SJ, Lennon SV, Bonham AM, Cotter TG. Induction of apoptosis
(programmed cell death) in human leukemic HL-60 cells by inhibition
of RNA or protein synthesis. J Immunol 1990;145:1859–67.
20. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue
of yeast Apg8p, is localized in autophagosome membranes after
processing. Embo J 2000;19:5720–8.
Cancer Res; 71(4) February 15, 2011
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
1429
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Drygin et al.
21. Zeng X, Kinsella TJ. Mammalian target of rapamycin and S6 kinase 1
positively regulate 6-thioguanine-induced autophagy. Cancer Res
2008;68:2384–90.
22. Collins RJ, Harmon BV, Souvlis T, Pope JH, Kerr JF. Effects of
cycloheximide on B-chronic lymphocytic leukaemic and normal lymphocytes in vitro: induction of apoptosis. Br J Cancer 1991;64:518–22.
23. Cheng Y, Li H, Ren X, Niu T, Hait WN, Yang J. Cytoprotective effect of
the elongation factor-2 kinase-mediated autophagy in breast cancer
cells subjected to growth factor inhibition. PLoS One 2010;5:e9715.
24. Young AR, Narita M, Ferreira M, et al. Autophagy mediates the mitotic
senescence transition. Genes Dev 2009;23:798–803.
25. Grummt I. Life on a planet of its own: regulation of RNA polymerase I
transcription in the nucleolus. Genes Dev 2003;17:1691–702.
26. Moss T. At the crossroads of growth control; making ribosomal RNA.
Curr Opin Genet Dev 2004;14:210–7.
27. Russell J, Zomerdijk JC. The RNA polymerase I transcription machinery. Biochem Soc Symp 2006:203–16.
28. Cai W, Hu L, Foulkes JG. Transcription-modulating drugs: mechanism
and selectivity. Curr Opin Biotechnol 1996;7:608–15.
29. Liebhaber SA, Wolf S, Schlessinger D. Differences in rRNA metabolism of primary and SV40-transformed human fibroblasts. Cell
1978;13:121–7.
30. Gorski JJ, Pathak S, Panov K, et al. A novel TBP-associated factor of
SL1 functions in RNA polymerase I transcription. Embo J 2007;26:
1560–8.
31. Denissov S, van Driel M, Voit R, et al. Identification of novel functional
TBP-binding sites and general factor repertoires. Embo J 2007;
26:944–54.
1430
Cancer Res; 71(4) February 15, 2011
32. Hannan KM, Hannan RD, Smith SD, Jefferson LS, Lun M, Rothblum LI.
Rb and p130 regulate RNA polymerase I transcription: Rb disrupts the
interaction between UBF and SL-1. Oncogene 2000;19:4988–99.
33. Zhai W, Comai L. Repression of RNA polymerase I transcription by the
tumor suppressor p53. Mol Cell Biol 2000;20:5930–8.
34. Zhang C, Comai L, Johnson DL. PTEN represses RNA Polymerase I
transcription by disrupting the SL1 complex. Mol Cell Biol 2005;25:
6899–911.
35. Fetherston J, Werner E, Patterson R. Processing of the external
transcribed spacer of murine rRNA and site of action of actinomycin
D. Nucleic Acids Res 1984;12:7187–98.
36. Muscarella DE, Rachlinski MK, Sotiriadis J, Bloom SE. Contribution
of gene-specific lesions, DNA-replication-associated damage, and
subsequent transcriptional inhibition in topoisomerase inhibitormediated apoptosis in lymphoma cells. Exp Cell Res 1998;238:
155–67.
37. Mischo HE, Hemmerich P, Grosse F, Zhang S. Actinomycin D induces
histone gamma-H2AX foci and complex formation of gamma-H2AX
with Ku70 and nuclear DNA helicase II. J Biol Chem 2005;280:9586–
94.
38. White E, DiPaola RS. The double-edged sword of autophagy modulation in cancer. Clin Cancer Res 2009;15:5308–16.
39. Chen N, Debnath J. Autophagy and tumorigenesis. FEBS Lett 2010;
584:1427–35.
40. Mahajan PB. Modulation of transcription of rRNA genes by rapamycin.
Int J Immunopharmacol 1994;16:711–21.
41. Sudarsanam S, Johnson DE. Functional consequences of mTOR
inhibition. Curr Opin Drug Discov Devel 2010;13:31–40.
Cancer Research
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.
Published OnlineFirst December 15, 2010; DOI: 10.1158/0008-5472.CAN-10-1728
Targeting RNA Polymerase I with an Oral Small Molecule CX-5461
Inhibits Ribosomal RNA Synthesis and Solid Tumor Growth
Denis Drygin, Amy Lin, Josh Bliesath, et al.
Cancer Res 2011;71:1418-1430. Published OnlineFirst December 15, 2010.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/0008-5472.CAN-10-1728
Access the most recent supplemental material at:
http://cancerres.aacrjournals.org/content/suppl/2016/06/16/0008-5472.CAN-10-1728.DC1
This article cites 39 articles, 16 of which you can access for free at:
http://cancerres.aacrjournals.org/content/71/4/1418.full.html#ref-list-1
This article has been cited by 44 HighWire-hosted articles. Access the articles at:
/content/71/4/1418.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected].
To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 2011 American Association for Cancer Research.