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BET Bromodomain Inhibitors Enhance Efficacy and Disrupt Resistance to AR Antagonists in the
Treatment of Prostate Cancer
Irfan A. Asangani1,2,10, Kari Wilder-Romans4, Vijaya L. Dommeti1, Pranathi M. Krishnamurthy1, Ingrid J. Apel1, June Escara-Wilke1, Stephen R. Plymate6, Nora M. Navone7, Shaomeng
Wang1,8,9, Felix Y. Feng1,4,9, Arul M. Chinnaiyan1,2,3,5,9*
1Michigan Center for Translational Pathology, Departments of 2Pathology, 4Radiation Oncology, 5Urology, 7Internal Medicine, Pharmacology, and Medicinal Chemistry, 3Howard Hughes Medical Institute, 9Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan. 6Department of Medicine, University of Washington and VAPSHCS, Seattle. 7Department of Genitourinary Medical Oncology, M. D. Anderson Cancer Center, Houston. 10Current Address: Department of Cancer Biology, University of Pennsylvania, Philadelphia, USA.
Corresponding Author: Arul M. Chinnaiyan, M.D., Ph.D., University of Michigan, 1400 E. Medical Center Dr., Ann Arbor, MI 48109-5940, USA. arul@umich.edu Disclosure of Potential Conflicts of Interest A.M.C. and S.W. are co-founders of Oncofusion Therapeutics, which is developing novel BET bromodomain inhibitors. I.A.A. has served as consultant to Oncofusion Therapeutics. Grant Support This work was supported by a Movember-Prostate Cancer Foundation Challenge Award and in part by the NCI Prostate SPORE (P50CA186786). A.M.C. is also supported by the American Cancer Society, and A. Alfred Taubman Institute. I.A.A. is supported by NIH pathway to independence (1K99CA187664-01) and a PCF Young Investigator Award.
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Abstract
Next generation anti-androgen therapies such as enzalutamide and abiraterone have had a profound impact on the management of metastatic castration-resistant prostate cancer (mCRPC). However, mCRPC patients invariably develop resistance to these agents. Here, a series of clonal cell lines were developed from enzalutamide-resistant prostate tumor xenografts to study the molecular mechanism of resistance and test their oncogenic potential under various treatment conditions. Androgen receptor (AR) signaling was maintained in these cell lines which acquired potential resistance mechanisms including expression of AR-variant 7 (AR-v7) and glucocorticoid receptor (GR). BET bromodomain inhibitors were shown previously to attenuate AR signaling in mCRPC; here, we demonstrate the efficacy of BET inhibitors in enzalutamide-resistant prostate cancer models. AR antagonists, enzalutamide and ARN509 exhibit enhanced prostate tumor growth inhibition when combined with BET inhibitors, JQ1 and OTX015, respectively. Taken together, these data provide a compelling pre-clinical rationale to combine BET inhibitors with AR antagonists to subvert resistance mechanisms. Implications: Therapeutic combinations of BET inhibitors and AR antagonists may enhance the clinical efficacy in the treatment of mCRPC.
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Introduction
Metastatic castration-resistant prostate cancer (mCRPC) leads to nearly 30,000 deaths
annually in the United States (1). AR is a major driver alteration in mCRPC (2-4) and most
tumors continue to rely on androgen receptor signaling (3). Second generation anti-androgen
therapies such as abiraterone and enzalutamide have become standard of care treatments for
mCRPC (5,6). However, most patients eventually develop resistance to these therapies and,
thus alternate approaches to target androgen signaling are needed.
Recently, our group and others have shown that bromodomain and extraterminal (BET)
inhibitors block mCRPC growth in animal models (7-9). BET inhibitors are epigenetic therapies
that target bromodomain containing proteins BRD2/3/4 and BRDT (10-12). They appear to
preferentially affect oncogenic transcription through a super-enhancer based mechanism
(13,14). mCRPC is a particularly attractive indication for BET inhibitors due to its reliance on
oncogenic transcription factor signaling mediated by AR, ETS fusions, and MYC (7). Several
groups including our own have shown that AR signaling is affected by BET inhibitors (7,8,15).
In the current study, we report the efficacy of BET inhibitors in enzalutamide-resistant
mCRPC models. We developed enzalutamide-resistant prostate cancer cells from murine
xenograft models in an attempt to understand the mechanisms of resistance to enzalutamide.
Clonal cell lines derived from distinct enzalutamide-resistant LNCaP-AR and VCAP tumors
displayed significantly higher AR expression and signaling relative to controls. Additionally,
enzalutamide-resistant CRPC cell lines maintained sensitivity to BET inhibitors, leading to
attenuation of AR signaling. Interestingly, AR-v7 which has been reported to be associated with
resistance to anti-androgen treatments (16), was specifically elevated in enzalutamide-resistant
VCaP cells and was markedly repressed by BET inhibitors. In addition to inhibiting the growth of
enzalutamide-resistant CRPC cell lines, BET inhibitors displayed enhanced efficacy in vivo
when combined with anti-androgens such as enzalutamide and ARN509.
Materials and methods
Murine Xenografts. Procedures involving mice were approved by the University Committee on
Use and Care of Animals at the University of Michigan and conform to all regulatory standards.
Please refer to Supplemental Materials and Methods for details.
Cell culture and Viability Assays. Prostate cancer cells were grown in ATCC recommended
media. Please refer to Supplemental Materials and Methods for details.
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Quantitative Real-Time PCR. Primer sequences are provided in the Table S1. RNA extraction,
cDNA synthesis and QPCRs were performed as previously described (7).
Western Blot Analyses. Antibodies used in the study are listed in Table S2. Western blot
analysis was performed as described previously (7).
Results and Discussion
The LNCaP-AR cell line was the key pre-clinical model used in the development of
enzalutamide where in castrated male mice, established LNCaP-AR xenograft tumors,
regressed upon enzalutamide treatment (17). In order to study the mechanism of resistance to
enzalutamide, we performed an in vivo drug efficacy experiment in castrated mice bearing
LNCaP-AR tumor xenografts. Consistent with previous studies (17,18), we initially observed
robust tumor regression in enzalutamide-treated animals compared to vehicle-treated controls
(Fig. 1A and B). However, after about 47days, the tumors became refractory to enzalutamide
treatment and tumor growth resumed. Next, we analyzed AR and AR target gene expression in
enzalutamide-resistant tumors; as shown in Fig. 1C and D, AR expression was elevated at the
transcript and protein levels as well as AR target genes compared to vehicle controls.
Interestingly, a few of the tumors displayed high levels of glucocorticoid receptor (GR) transcript
and protein expression as reported earlier (18). However, we did not observe high AR
signaling, as measured by AR target gene expression, in the GR-overexpressing tumors (Fig. 1C, green boxes- tumor with GR outlier).
Furthermore, to expand the model systems used to study the mechanisms of resistance
to enzalutamide, we conducted a murine xenograft experiment using VCaP prostate cancer
cells that harbor the TMPRSS2:ERG gene fusion and AR amplification, both of which are
frequent molecular aberrations in patients with advanced mCRPC. Treatment of VCaP tumor-
bearing mice with enzalutamide (30mg/kg) for 5days/week over 5weeks led to a small but
significant reduction in tumor volume compared to vehicle controls (Supplementary Fig.1A).
Further, AR and AR-v7 expression levels were elevated in the enzalutamide-treated tumors
relative to vehicle controls (Supplementary Fig.1B), consistent with a previous report that
found elevated AR-v7 expression in circulating tumor cells from mCRPC patients with acquired
resistance to enzalutamide and abiraterone treatment (16). Additionally, AR downstream targets
such as SLC45A3 and MYC were also upregulated in enzalutamide-treated VCaP tumors.
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To evaluate the efficacy of BET inhibitors in enzalutamide-refractory disease, we
established multiple cell lines from enzalutamide-resistant LNCaP-AR and VCaP tumors that
were harvested and cultured in vitro in the presence of 5uM enzalutamide (Fig. 2A and B). In
LNCaP-AR enzalutamide-resistant cells, all clones expressed varying levels of AR and PSA
while only 1 out 5 clones displayed high GR expression (Fig. 2A). In VCaP enzalutamide-
resistant cell lines we observed a significant increase in full length AR and AR-v7 levels with
active AR signaling in all clones (Fig. 2B and Supplementary Fig. 1).
As these tumor derived cell line clones are enzalutamide-resistant, we asked whether
these cells would respond to a BET inhibitor. We first directly compared the efficacies of three
different BET inhibitors, JQ1, OTX-015 and I-BET762 in LNCaP vs LNCaP-AR cells and found
the first two compounds to be equally efficacious in LNCaP-AR cells with an IC50 value of
approximately 65nM, whereas parental LNCaP cells displayed lower sensitivity to the
compounds with an IC50 value of approximately 110nM (Supplementary Fig. 2A and B); we
chose JQ1 as the representative BET inhibitor in subsequent studies. We treated 3 independent
enzalutamide-resistant LNCAP-AR sub-lines with varying concentrations of JQ1 and analyzed
for cell viability 4days post-treatment, and found all 3 sub-lines were sensitive to JQ1 with IC50
values of approximately 100nM (Fig. 2C). In order to evaluate the long term effects of JQ1
treatment in the presence of enzalutamide, we performed colony-formation assays. As
expected, parental LNCaP-AR cells were sensitive whereas the enzalutamide-resistant clones
were insensitive to enzalutamide treatment (Fig. 2D). However, even at low concentrations
(100nM) of JQ1, proliferation of enzalutamide-resistant sub-lines were severely inhibited in the
long term (Fig. 2D), demonstrating that these AR signaling-positive cells are inherently
susceptible to BET inhibition.
In parallel experiments, we tested enzalutamide-resistant VCaP derivatives for sensitivity
to BET inhibition; 3 independent enzalutamide-resistant VCaP sub-lines were treated with
varying concentrations of JQ1 and analyzed for cell viability 4days post-treatment. All 3 resistant
sub-lines along with parental VCaP cells displayed sensitivity to JQ1 with IC50 values of less
than 100nM (Fig. 2E). As expected, VCaP parental cells were sensitive to enzalutamide in long
term colony formation assays. While enzalutamide-resistant VCaP sub-lines were insensitive to
enzalutamide, these cells were sensitive upon JQ1 treatment in long term colony formation
assay (Fig. 2F), demonstrating that these AR-amplified, AR-v7-overexpressing cell lines are
also susceptible to BET inhibition.
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As enzalutamide-resistant clones showed an increase in AR levels but nonetheless
remained sensitive to BET inhibitors, we examined whether BET inhibitors would have an effect
on AR-mediated gene expression in these cells. Three independent enzalutamide-resistant
LNCaP-AR sub-lines were treated with vehicle or two different concentrations of JQ1 and
expression of AR target genes was analyzed by qRT-PCR and western blot analysis. As shown
in Fig. 3A, FKBP5, KLK3, TMPRSS2 and MYC were transcriptionally down-regulated upon JQ1
treatment in the parental LNCaP-AR as well as in all three enzalutamide-resistant sub-lines.
Similarly, PSA and MYC proteins that were expressed in the presence of enzalutamide in the
resistant sub-lines were significantly repressed upon JQ1 treatment (Fig. 3B). Interestingly, AR
protein levels were elevated with increasing doses of JQ1 in the parental LNCaP-AR and
resistant sub-lines (Fig. 3B), suggesting perturbation of a negative feedback loop controlling AR
levels.
Similarly, we sought to determine the effect of JQ1 on AR signaling and in particular,
AR-v7 expression in enzalutamide-resistant VCaP sub-lines. Parental VCaP and 3 independent
enzalutamide-resistant sub-lines described above were treated with JQ1 followed by qRT-PCR
and western blot analysis of AR target genes. FKBP5, KLK3, ERG and MYC were
transcriptionally down-regulated upon JQ1 treatment (Fig. 3C); ERG, MYC and PSA protein
levels were also reduced (Fig. 3D). Surprisingly, AR-variant protein levels were down-regulated
but not full length AR in all 3 resistant sub-lines upon JQ1 treatment (Fig. 3D, top panel); AR-
variant was confirmed to be AR-v7 by AR-v7-specific antibody (Fig. 3E). Splicing factors,
SRSF1 and U2AF65, reported to be involved in the generation of AR splice variants (19), are
subject to down-regulation by JQ1 and would explain the down-regulation of AR-v7 observed
here (Fig. 3F). Interestingly, the SRSF1 and U2AF65 promoter regions are enriched for
BRD2/3/4 protein displayed reduced levels upon JQ1 treatment (Supplementary Fig. 3). These
data suggest that the anti-proliferative effect of JQ1 in the enzalutamide-resistant cells is partly
due to its inhibitory effect on AR-v7 generation, which has been proposed as one of the major
drivers of resistance to androgen deprivation therapy (ADT) (16).
To extend upon the in vitro studies above, we next sought to conduct BET inhibition
studies in vivo using the castrated VCaP xenograft mouse model (7). Although VCaP cells are
originally derived from a patient with CRPC (20), these cells require androgen supplementation
for growth in culture and therefore VCaP tumor xenografts are castration-responsive in mouse
models (21). Castrated VCaP-tumor bearing mice were maintained until the tumors reached
their original pre-castration volume and then treated with JQ1 (50mg/kg/day), enzalutamide
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(10mg/kg/day) or a combination. As previously observed (7), JQ1 alone led to approximately
45% reduction in the castration-resistant VCaP tumors compared to vehicle controls at 30days
post treatment. However, when JQ1 was combined with enzalutamide tumor growth was
inhibited by 62% (Fig. 4A). Further, we assessed the doubling of tumor volume (time interval
between initiation of treatment to tumor progression) by generating Kaplan-Meier survival
curves and compared the treatment groups using long-rank test. Tumor progression was
delayed for the JQ1-treated group (p=0.0008) with a median tumor doubling of 15days whereas
tumor doubling for vehicle treated mice was 10.5days, with no significant difference in the
enzalutamide alone treatment group (12days; p=0.215). However, the combination treatment
group displayed a marked delay in tumor progression (p<0.0001) with a median doubling of 22
days (Fig. 4B). The qRT-PCR analysis of tumors from the various treatment groups showed
robust silencing of driver oncogenes, AR-v7, ERG, and MYC in mice treated with JQ1 alone or
in combination with enzalutamide whereas treatment with enzalutamide alone showed no
change or an increase in AR and AR-v7 expression respectively compared to vehicle controls
(Fig. 4C). Additionally, in an AR- and ERG-positive patient-derived xenograft model we
observed greater anti-tumor efficacy of JQ1 and enzalutamide in combination than JQ1 alone
(Supplementary Fig. 4). Interestingly, JQ1 treatment did not show anti-tumor activity in AR and
ERG-negative patient derived xenografts.
Next, we tested the in vivo efficacy of OTX-015, a BET inhibitor that is being evaluated in
a phase Ib clinical trial that includes metastatic prostate cancer, alone or in combination with
ARN-509, a second generation anti-androgen related to enzalutamide (22), in the castrated
VCaP xenograft mouse model. As shown in Fig. 4D, treatment with a combination of OTX-015
and ARN-509 led to robust anti-tumor activity resulting in 82% growth inhibition in mice, in
contrast to 53% and 60% inhibition in mice treated with ARN509 or OTX-015 alone respectively.
Likewise, the progression-free survival as measured by tumor doubling was significantly
increased for all the treatment groups (P<0.0001) with median doubling of 21 and 21.5 days for
ARN509 and OTX-015 respectively compared to 7 days for vehicle-treated group. The median
doubling time for the combination treatment group could not be determined over the course of
the experiment as the group did not achieve threshold progression (Fig. 4E). Interestingly, ARN-
509 alone had improved anti-tumor activity in this model than enzalutamide as was observed in
LNCaP-AR model by Clegg et al (23). Furthermore, gene expression analysis of tumors from
the OTX-015 and ARN-509 combination treatment groups showed robust silencing of AR-v7,
ERG, and MYC while AR and AR-v7 levels were elevated in tumors from mice treated with
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ARN-509 alone compared to vehicle controls (Fig.4F). These in vivo data demonstrate
enhanced efficacy of combined BET inhibitors and anti-androgens.
Multiple paths leading to primary or acquired resistance to anti-androgen therapies have
been reported including overexpression of androgen synthesis enzymes, amplification and
point mutations in AR, overexpression of AR splice variants and induction of GR (16,18,24,25).
Our cell line and in vivo mouse data recapitulated several of these paths to resistance to
enzalutamide and we demonstrated that BET inhibitors could overcome this resistance. The
BET inhibitor efficacy data presented here has implications on the design of subsequent clinical
trials that will most likely follow the ongoing multiple phase 1 trial of BET inhibitors in castration
resistant prostate cancer (www.clinicaltrials.gov). We anticipate that combining BET inhibitors
with second generation anti-androgens such as enzalutamide or ARN-509 will result in more
durable therapeutic responses.
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Figure Legends
Figure 1. Active AR signaling in enzalutamide-resistant xenograft tumors. A, LNCaP-AR
cells were implanted subcutaneously in castrated mice and grown until tumors reached a size of
approximately 100mm3. Xenografted mice were randomized and received vehicle or 10mg/kg
enzalutamide, 5days a week. Mean tumor volume ±s.d. is shown. B, Individual tumors from A
are shown. C, RT-qPCR analysis of AR and AR target genes in LNCaP-AR xenograft tumors
Blue box with green boundary indicates tumor sample with outlier GR expression. D, Western
blot analysis in tumor. Parental LNCaP-AR and 22RV1 were positive controls. Note tumor
sample 973L with outlier GR protein and corresponding PSA levels. The sample number
denotes the animal ID.
Figure 2. Enzalutamide resistant tumor-derived cells maintain BETi sensitivity. A, Western
blot analysis with lysates from enzalutamide-resistant tumor-derived LNCaP-AR cell lines. B,
Western blot analysis with lysates from enzalutamide-treated tumor-derived VCaP cell lines
(n=8). VCaP cells treated with DMSO or 5μM enzalutamide served as control. Note the
overexpression of full-length AR and AR-variant in enzalutamide-resistant VCaP derivatives. C,
Cell viability curves for three independent enzalutamide-resistant LNCaP-AR cells treated with
JQ1. Crystal violet imaging of the 96 well plate is shown. D, Colony formation assay; cells were
cultured in the presence or absence of drugs as indicated for 14days followed by staining.
Quantification is shown at bottom right. E, Cell viability curves for three independent
enzalutamide-resistant VCaP cancer cells treated with JQ1. Parental VCaP cells served as
control. F, Colony formation assay; cells were cultured in the presence or absence of drugs as
indicated for 14days followed by imaging. Quantification (relative number of cells) is shown at
bottom right.
Figure 3. BETi blocks AR signaling in enzalutamide resistant cells. A, RT-qPCR analysis of
AR target genes in three independent LNCaP-AR ERTCs grown in 5uM enzalutamide, treated
with JQ1 for 24hrs. Parental LNCaP-AR cells were treated with either enzalutamide alone and in
combination with JQ1. Fold mRNA expression relative to the respective DMSO control is shown.
The values are mean of 3 independent readings. B, Western blot analysis of indicated target
proteins in the cells from A treated with JQ1 for 48hrs. Cleaved-PARP antibody was used to
detect apoptosis. C, As in A with parental VCaP and three independent enzalutamide-resistant
VCaP lines. D, As in B. Note the loss of AR-variant band but not the full-length AR band upon
JQ1 treatment. TDRD1 is a direct target of ERG and was used as a control for ERG function.
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Star symbol - non-specific band. E, Western blot with AR-v7 specific antibody using the cell
lysates from the same experiment. F, As in C with target specific primers.
Figure 4. BETi in combination with enzalutamide or ARN-509 demonstrates enhanced anti-tumor activity: A, Castrated mice bearing VCaP CRPC xenograft received vehicle or
10mg/kg enzalutamide or 50mg/kg JQ1 or enza./JQ1 combination as indicated 5days/week.
Percentage tumor volume ±s.e.m. is shown. Statistical significance by two-tailed Student’s t-test
* P=0.012; ** P<0.0001; *** P=0.005. B, The cumulative incidence plot depicting the percentage
of tumors in each treatment group that have doubled in volume as a function of time. C, RT-
qPCR analysis of indicated target gene expression in xenograft tumors. Relative fold expression
with mean ±s.e.m is shown. D, As in A, except with 10mg/kg ARN-509 or 100mg/kg OTX-015 or
ARN-509/OTX-015 combination. Percentage tumor volume ±s.e.m. is shown. Statistical
significance by two-tailed Student’s t-test. * P=0.0045; ** P<0.001; *** P=0.0001, P=0.007. E,
As in B. F, As in C.
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Acknowledgments
We thank A. Palanisamy for technical assistance; M. Cieslik and R. Malik for helpful
discussions; J. Athanikar and K. Giles for critically reading the manuscript and submission of
documents.
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(pe
rce
nta
ge
)
Days of drug treatment Days of drug treatment
Vehicle (n=9)
Enzalutamide (n=9)
A B
C
D
LNC
aP-A
R
22R
V1
vehicle control
tumors
Enzalutamide
resistant tumors
AR
PSA (long exposure)
GAPDH
GR (short exposure)
Castrated LNCaP-AR xenograft model
Enzalutamide resistant tumorsVehicle control tumors
K LK 3
0
2
4
6
8
1 0
0
2
4
6
8
AR
Fo
ld m
RN
A e
xp
ressio
n
FKBP5
0
2
4
6
8
1 0
SLC45A3
0
5
10
15
NDRG1
0
2
4
6
PSA (short exposure)
0
40
80
120
GR
GR (long exposure)
MYC
0
1
2
3
4
908R
908L
947L
954R
954L
957L
957R
965R
965L
973R
973L
907R
909R
910R
926R
950R
on May 26, 2020. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 20, 2016; DOI: 10.1158/1541-7786.MCR-15-0472
Figure 2
908R (IC50 116nM)
957L (IC50 99nM)
973L (IC50 110nM)
908R
957L
973L
Ce
ll via
bili
ty
50
100
0.01 0.1 101
JQ1 (µM)
C
BC
ell
via
bili
ty
50
100
0.01 0.1 101
JQ1 (µM)
VCaP (IC50 90nM)
325R (IC50 48nM)
345R (IC50 62nM)
380R (IC50 75nM)
VCaP
VCaP
ERTC
325R
VCaP
ERTC
380R
VCaP
ERTC
345R
Enzalutamide (5µM)
- 100nM 500nM JQ1
E
D
100 42 6 2
100 10 2
100 6 1
100 4 0
AR
PSA
GAPDH
GR
LNC
aP-A
R
LNC
aP
VC
aP
908R
957L
965L
973R
973L
Enzalutamide
resistant tumor derived
cell lines (ERTC- 5µM)
A
AR
ERG
PSA (long exposure)
ACTIN
DM
SO
5µ
M E
nz.
325
R
325
L 3
45R
345
L
380
R 3
80L
387
R
387
L
PSA (short exposure)
AR-variant
VCaP - Enzalutamide
resistant tumor derived
cell lines (ERTC- 5µM)
F
AR-v7
AR (long exposure)
AR-variant
10 4
LNCaP-AR
LNCaP-AR
ERTC 973L
LNCaP-AR
ERTC 908R
LNCaP-AR
ERTC 957L
Enzalutamide (5µM)
- 100nM 500nM JQ1
100 44 21 1
100 21 1
22 4100
100
on May 26, 2020. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 20, 2016; DOI: 10.1158/1541-7786.MCR-15-0472
Figure 3
VCaP 325R 345R 380R
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
AR
PSA
MYC
cleaved-PARP
ACTIN
ERG
TDRD1
(ERG target)*
AR-variant
: JQ1
cleaved-PARP
AR
MYC
PSA
ACTIN
LNCaP-AR 908R 957L 973L
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM : JQ1
VCaP
325R
345R
380R
FKBP5
KLK3
ER
G
MYC
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
En
za
luta
mid
e (5
µM
)
LNCaP-AR
908R
957L
973L
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
: JQ1
En
za
luta
mid
e (5
µM
)
A B
C D
KLK3
FKBP5
TM
PR
SS2
MYC
VCaP 325R 345R 380R
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM
Enzalutamide (5µM)
- 0.5µM 2.5µM : JQ1
AR-v7
ACTIN
AR
-v7
SR
SF1
U2AF65
VCaP
325R
345R
380R
: JQ1
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
-
0.5µM
2.5µM
En
za
luta
mid
e (5
µM
)
: JQ1
Fold mRNA expression:
Fold mRNA expression:
Fold mRNA expression:E F
hnRNP1
1.00 1.00 1.00 1.00
0.44 0.52 0.89 1.40
0.43 0.03 0.60 0.21
0.42 0.00 0.29 0.09
1.06 1.00 1.00 1.00
0.23 0.02 1.06 0.19
0.24 0.00 0.68 0.09
1.01 1.00 1.00 1.00
0.17 0.02 1.26 0.25
0.16 0.00 0.84 0.14
1.07 1.00 1.00 1.00
0.24 0.02 1.18 0.24
0.22 0.00 0.43 0.08
1.01 1.05 0.96 1.02
0.95 0.82 0.85 1.03
0.56 0.02 0.10 0.08
0.46 0.02 0.05 0.04
0.95 0.99 1.03 1.05
0.11 0.00 0.36 0.03
0.13 0.00 0.14 0.03
1.01 1.03 0.99 1.01
0.72 0.04 1.01 0.34
0.18 0.04 0.43 0.06
1.01 1.00 0.99 1.06
0.36 0.00 0.81 0.16
0.27 0.00 0.27 0.05
1.00 1.00 1.00
4.60 1.21 1.04
1.29 0.81 1.44
1.07 0.47 0.74
1.00 0.97 1.02
0.24 0.55 0.74
0.16 0.50 0.56
1.00 1.01 1.06
0.16 0.57 0.87
0.12 0.43 0.81
1.00 0.98 1.10
0.20 0.44 0.90
0.16 0.23 0.47on May 26, 2020. © 2016 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from
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Figure 4
Vehicle
Enzalutamide (10mg/kg)
JQ1 (50mg/kg)
JQ1+Enzalutamide
A B
1 7 1 4 2 0 2 4 2 7 3 0
0
1 00
2 00
3 00
4 00
5 00
6 00
Days of drug treatment
Pe
rce
nta
ge
tu
mo
r vo
lum
e
**
Castrated VCaP xenograft model
D
D ay s0 1 0 2 0 3 0
0
5 0
1 00
Vehicle
Enzalutamide
JQ1
JQ1+Enzalutamide
Pro
gre
ssio
n fre
e s
urv
iva
l
1 3 6 8 1 0 1 3 1 6 2 0 2 3 2 7 3 0
0
1 00
2 00
3 00
4 00
5 00
6 00
7 00V eh ic le
A RN 50 9 10 mg /kg
O TX 01 5 10 0m g/kg
A RN +O TX
Pe
rce
nta
ge
tu
mo
r vo
lum
e
0 1 0 2 0 3 0
0
5 0
1 00
V eh ic le
A RN 50 9
O TX 01 5
A RN +O TX
Castrated VCaP xenograft model
D ay sDays of drug treatment
**
**
**
E
*
**
***
***
Pro
gre
ssio
n fre
e s
urv
iva
l
C AR-v7
Fo
ld m
RN
A e
xp
ressio
n
AR
0
1
2
3
4 P = 0.06
P =0.01P < 0.0001
P = 0.001
P < 0.0001
0
5
1 0
1 5 P < 0.0001
E RG
0 .0
0 .5
1 .0
1 .5
2 .0 P = 0.0002
P = 0.60P = 0.002
M YC
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
P = 0.6
P < 0.0001P < 0.0001
Vehicle EnzalutamideJQ1 JQ1+Enzalutamide
0
2
4
6
0
2
4
6
8
AR AR-v7
Fo
ld m
RN
A e
xp
ressio
n
P = 0.001
P = 0.0002P = 0.0001
P <0.0001
P <0.0001P <0.0001
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
E RG
P<0.0001
P = 0.50P<0.0001
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
M YC
P<0.0001
P = 0.01
P<0.0001
Vehicle A RN 50 9 O TX 01 5 A RN +O TX
F
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Published OnlineFirst January 20, 2016.Mol Cancer Res Irfan A. Asangani, Kari Wilder-Romans, Vijaya L. Dommeti, et al. CancerResistance to AR Antagonists in the Treatment of Prostate BET Bromodomain Inhibitors Enhance Efficacy and Disrupt
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