# Synergism of Heat Shock Protein 90 and Histone Deacetylase Inhibitors in Synovial Sarcoma

Authors: Anne Nguyen; Le Su; Belinda Campbell; Neal M. Poulin; Torsten O. Nielsen
Journal: Sarcoma
 (2009)

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## Abstract

Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG  have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of
17-AAG  with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only  induce cell death but also activate the survival pathway NF-κB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-κB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-κB activation. In our assays, adding 17-AAG blocks NF-κB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-κB inhibitor  BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-κB synergize with HDAC inhibitors against synovial sarcoma.


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## Body

## 1. Introduction

Synovial sarcoma is an aggressive malignancy, typically
occurring in the soft tissues of young adults, comprising up to 10% of adult
sarcomas in some series [1]. Current therapies for synovial sarcoma involve
surgical resection followed by either radiotherapy and/or chemotherapy (such as
doxorubicin) [2], with 5-year metastasis-free survival rates between 48% and 68%
[3–5]. The chromosomal translocation t(X;18)(p11.2;q11.2), resulting in an
SYT-SSX fusion oncoprotein, is demonstrable in almost all cases [1], but its molecular function is unclear, and it
is not directly targeted by established drugs.The heat shock protein 90 (Hsp90) inhibitor 17-(allylamino)-17-demethoxygeldanamycin
(17-AAG) is effective against synovial sarcoma in vitro [6]. Hsp90 is a chaperone assisting in the folding of
client proteins such as CDK-4, Raf-1 [7], HER2 [8], mutant p53 [9], Bcr-Abl [10],
IKK [11], and RIP [12] which play important roles in oncogenic cell growth,
division, and survival. Hsp90 inhibition results in degradation of its client
proteins, inhibition of tumor growth, induction of differentiation and
activation of apoptosis [13]. The Hsp90 inhibitor 17-AAG has completed phase I
clinical trials [14] and is in phase II for several malignancies including
metastatic melanoma, breast cancer, and ovarian cancer [15].The HDAC inhibitor romidepsin (FK228; depsipeptide) has also been
demonstrated to be effective against synovial sarcoma models, where nanomolar
levels cause histone acetylation, inhibition of growth, and invasion in cell
cultures and mouse xenografts [16]. We have confirmed these findings using the
HDAC inhibitors trichostatin A, romidepsin, and MS-275 on synovial sarcoma cell lines [17, 18]. HDAC
activity acts within transcription factor complexes to suppress transcription
at target loci, including tumor suppressors and genes driving differentiation [19, 20] by decreasing net acetylation of histones. HDAC inhibitors lead to
acetylation of several other proteins, including p53 [21], NF-κB [22], and Hsp90 [23] which have roles in
cellular growth, survival, and protein folding. Several HDAC inhibitors
(including MS-275; an orally bioavailable agent) are currently involved in
phase I and II clinical trials of malignancies including leukemias, lymphomas,
melanomas, and refractory solid tumors [24]. In synovial sarcoma, Ito et al.
have shown that the SYT partner of the SYT-SSX fusion oncoprotein interacts
with a HDAC complex, providing a mechanism for specific activity of HDAC
inhibitors in this disease [25]. Furthermore, we have recently shown that HDAC
inhibitor action reverses polycomb-mediated epigenetic suppression of SYT-SSX
target genes [26].The evidence that both 17-AAG and HDAC inhibitors are
individually effective against synovial sarcoma raises the question of whether
combinations would be synergistic. To date, synergy studies using Hsp90
inhibitors and various HDAC inhibitors (SAHA, sodium butyrate, and cinnamic
hydroxamic acid analog) have shown positive results in variety of human leukemia
cells [10, 27, 28]. However, in other models, the combination appears
antagonistic; Huang et al. reported that pretreatment with the Hsp90 inhibitor
geldanamycin before addition of trichostatin A averted death in COS-7 cells [29], and Yang et al. found that coadministration
of romidepsin with
17-AAG had neither synergistic nor additive effects on RUNX1-ETO levels in
Kasumi-1 leukemic cells [30]. As each type of agents is individually effective
against synovial sarcoma cells, and synergy could potentially be of great
benefit to patients, in this study, we seek to test combinations of 17-AAG with
the HDAC inhibitor MS-275 for efficacy against synovial sarcoma.A possible mechanism for synergy
between HDAC and Hsp90 inhibitors involves effects on the survival pathway NF-κB. NF-κB is a transcription factor constitutively
activated in many cancer models [31], wherein it confers resistance to
apoptosis and promotes cell survival [32], angiogenesis, and invasion [33].
Expression profiling studies by us and others have shown that RIPK4, an activator of NF-κB [34], is highly expressed within synovial
sarcoma primary tumor samples [35]. HDAC3 regulates acetylation of the NF-κB subunit RelA, thereby reducing its
transcriptional activity [22]. Other studies have confirmed that HDAC
inhibitors induce NF-κB activity;
an effect which diminishes the lethality of these drugs against lung cancer
cell lines [36]. In contrast, 17-AAG has been shown to be an effective
inhibitor of the NF-κB pathway [12].
In this work, we also investigate if 17-AAG can synergize with HDAC inhibitors
by reducing the activation of NF-κB.


## 2. Materials and Methods

### 2.1. Reagents

17-AAG was
provided under the terms of a materials transfer agreement (MTA) with the
Developmental Therapeutics Branch of the National Cancer Institute
(Bethesda, Md, USA) through Kosan Biosciences (Hayward, Calif, USA). MS-275 was
provided under the terms of an MTA by Schering AG (Berlin, Germany) through Berlex Pharmaceuticals
(Montville, NJ, USA). RPMI 1640, fetal bovine serum, and trypsin were purchased
from Life Technologies (Invitrogen, Mississauga, ON, Canada). siRNA
targeting HDAC3 was obtained
from Santa Cruz Biotechnology (Santa Cruz, Calif, USA), catalog number
sc-35538; a 50 ng dose was confirmed by western blot to knock down HDAC3 in
synovial sarcoma cells by 71% at 24 hours relative to scrambled siRNA
control.  BAY 11-7085 was purchased from
Calbiochem (San Diego, Calif, USA). The pNF-κB-Luc plasmid, containing the firefly
luciferase (luc) gene from photinus pyralis and multiple copies of
the NF-κB
consensus sequence fused to a TATA-like promoter region from the herpes simplex
virus thymidine kinase promoter, was from Clontech (Mountain View, Calif, USA).


### 2.2. Monolayer Cell Culture and Drug Effect Assays

The biphasic synovial sarcoma cell
line SYO-1 and the monophasic human cell line Fuji were kindly provided by
Akira Kawai (National Cancer Centre Hospital, Tokyo, Japan), and
Kazuo Nagashima (Hokkaido University School of Medicine, Sapporo, Japan),
respectively [37, 38].  MTT proliferation and annexin
V-FITC/propidium iodide flow cytometry assays were performed as previously
described [6].


### 2.3. Protein Quantification

Sample protein concentrations were
determined by bicinchoninic acid assay (BCA Protein Assay kit,
Pierce, Rockford, Ill, USA) as per manufacturer’s instructions. Samples were
measured for absorbance at 562 nm in a PowerWaveX enzyme-linked immunoabsorbent
assay plate reader from Bio-Tek Instruments (Winooski, Vt, USA).


### 2.4. Lysate Preparation

Total cellular extracts were
prepared in lysis buffer (10 mM Tris pH 7.5, 1 mM EGTA, 150 mM NaCl, 1% Triton
X-100, 0.5% Nonidet P-40, 1 mM Na3VO4, and 1 mM PMSF) from
4  × 105 cells, incubated on ice for 20 minutes, and centrifuged at
10 000 g to remove cellular debris. Purified nuclear and cytoplasmic extracts
were prepared by using the NE-PER Nuclear and Cytoplasmic Extraction Reagents
kit (Pierce, Rockford, Ill, USA).


### 2.5. Immunoblot Analysis

Mouseα-acetyl lysine and rabbit α-p65 were purchased from Abcam (Cambridge, Mass,
USA), mouse α-IκBα from Cell
Signaling (Beverly, Mass, USA), and rabbit α-p85 from Upstate Millipore (Charlottesville, Va,
USA). Goat α-rabbit
HRP and goat α-mouse HRP
secondary antibodies were purchased from Pierce Biotechnology (Rockford, Ill,
USA).  Protein samples were loaded onto a
10% SDS polyacrylamide gel. Western blotting of the samples was done according
to standard procedures. Membranes were incubated in 1 mL of SuperSignal West Femto
Luminol/Enhancer Solution with 1 mL Stable Peroxide Buffer from Pierce (Rockford, Ill,
USA) at room temperature for 2 minutes and exposed onto photographic film.


### 2.6. Luciferase Assays

SYO-1 cells were plated onto 24-well
plates at 4× 104 cells/well. SYO-1 cells were transfected with 0.3 μg of plasmid/well using FuGENE 6 Transfection
reagent (Roche Applied Science, Indianapolis, Ind, USA) as per manufacturer’s
instructions, and they were treated the following day. After 24 hours of treatment,
cells were washed and 100 μL of ice
cold 1× Passive Lysis Buffer from the Dual-Luciferase Reporter Assay System kit
(Promega, Madison, Wis, USA) was added. Cells were incubated shaking for 20
minutes at room temperature. Samples were aliquoted 3 times at 20 μL/well to a luciferase plate and twice at 10 μL/well to a 96-well plate for protein quantification.
Samples were injected with 50 μL/well of
LARII from the kit and read on an EG&G Berthold microplate luminometer 96 V
(Germany). Samples were normalized to total protein concentration, and average
values were compared to vehicle control set to 1.


### 2.7. Synergism Analysis

Synergy was quantified according to
the protocol published by Chou and Talalay [39]. The log of the fraction
affected/fraction unaffected was plotted as a function of the log of the dose
to determine the IC50 from the equation log(fa/fu) = mlog(D)  −mlog (IC50).
Dose responses of drugs in combination were tested at a fixed ratio.
Combination index (CI) values were obtained from the equation CIx = Dc1x/D1x +
Dc2x/D2x + Dc1xDc2x/D1xD2x, where Dc1x is dose of drug 1 in combination
required for achieving x percent of cell inviability. Combination index values below 1 are
indication of synergism, near 1 of additivity, and greater than 1 of
antagonism. MTT and NF-κB luciferase
reporter experiments using the drugs as single agents and in combination were
done in triplicates, and all experiments to determine synergism were repeated
at least once. Annexin V-FITC apoptosis assays and western blotting assays were
likewise repeated once. Statistical analyses on replicates were performed by calculating 95% confidence
intervals.


## 2.1. Reagents

17-AAG was
provided under the terms of a materials transfer agreement (MTA) with the
Developmental Therapeutics Branch of the National Cancer Institute
(Bethesda, Md, USA) through Kosan Biosciences (Hayward, Calif, USA). MS-275 was
provided under the terms of an MTA by Schering AG (Berlin, Germany) through Berlex Pharmaceuticals
(Montville, NJ, USA). RPMI 1640, fetal bovine serum, and trypsin were purchased
from Life Technologies (Invitrogen, Mississauga, ON, Canada). siRNA
targeting HDAC3 was obtained
from Santa Cruz Biotechnology (Santa Cruz, Calif, USA), catalog number
sc-35538; a 50 ng dose was confirmed by western blot to knock down HDAC3 in
synovial sarcoma cells by 71% at 24 hours relative to scrambled siRNA
control.  BAY 11-7085 was purchased from
Calbiochem (San Diego, Calif, USA). The pNF-κB-Luc plasmid, containing the firefly
luciferase (luc) gene from photinus pyralis and multiple copies of
the NF-κB
consensus sequence fused to a TATA-like promoter region from the herpes simplex
virus thymidine kinase promoter, was from Clontech (Mountain View, Calif, USA).


## 2.2. Monolayer Cell Culture and Drug Effect Assays

The biphasic synovial sarcoma cell
line SYO-1 and the monophasic human cell line Fuji were kindly provided by
Akira Kawai (National Cancer Centre Hospital, Tokyo, Japan), and
Kazuo Nagashima (Hokkaido University School of Medicine, Sapporo, Japan),
respectively [37, 38].  MTT proliferation and annexin
V-FITC/propidium iodide flow cytometry assays were performed as previously
described [6].


## 2.3. Protein Quantification

Sample protein concentrations were
determined by bicinchoninic acid assay (BCA Protein Assay kit,
Pierce, Rockford, Ill, USA) as per manufacturer’s instructions. Samples were
measured for absorbance at 562 nm in a PowerWaveX enzyme-linked immunoabsorbent
assay plate reader from Bio-Tek Instruments (Winooski, Vt, USA).


## 2.4. Lysate Preparation

Total cellular extracts were
prepared in lysis buffer (10 mM Tris pH 7.5, 1 mM EGTA, 150 mM NaCl, 1% Triton
X-100, 0.5% Nonidet P-40, 1 mM Na3VO4, and 1 mM PMSF) from
4  × 105 cells, incubated on ice for 20 minutes, and centrifuged at
10 000 g to remove cellular debris. Purified nuclear and cytoplasmic extracts
were prepared by using the NE-PER Nuclear and Cytoplasmic Extraction Reagents
kit (Pierce, Rockford, Ill, USA).


## 2.5. Immunoblot Analysis

Mouseα-acetyl lysine and rabbit α-p65 were purchased from Abcam (Cambridge, Mass,
USA), mouse α-IκBα from Cell
Signaling (Beverly, Mass, USA), and rabbit α-p85 from Upstate Millipore (Charlottesville, Va,
USA). Goat α-rabbit
HRP and goat α-mouse HRP
secondary antibodies were purchased from Pierce Biotechnology (Rockford, Ill,
USA).  Protein samples were loaded onto a
10% SDS polyacrylamide gel. Western blotting of the samples was done according
to standard procedures. Membranes were incubated in 1 mL of SuperSignal West Femto
Luminol/Enhancer Solution with 1 mL Stable Peroxide Buffer from Pierce (Rockford, Ill,
USA) at room temperature for 2 minutes and exposed onto photographic film.


## 2.6. Luciferase Assays

SYO-1 cells were plated onto 24-well
plates at 4× 104 cells/well. SYO-1 cells were transfected with 0.3 μg of plasmid/well using FuGENE 6 Transfection
reagent (Roche Applied Science, Indianapolis, Ind, USA) as per manufacturer’s
instructions, and they were treated the following day. After 24 hours of treatment,
cells were washed and 100 μL of ice
cold 1× Passive Lysis Buffer from the Dual-Luciferase Reporter Assay System kit
(Promega, Madison, Wis, USA) was added. Cells were incubated shaking for 20
minutes at room temperature. Samples were aliquoted 3 times at 20 μL/well to a luciferase plate and twice at 10 μL/well to a 96-well plate for protein quantification.
Samples were injected with 50 μL/well of
LARII from the kit and read on an EG&G Berthold microplate luminometer 96 V
(Germany). Samples were normalized to total protein concentration, and average
values were compared to vehicle control set to 1.


## 2.7. Synergism Analysis

Synergy was quantified according to
the protocol published by Chou and Talalay [39]. The log of the fraction
affected/fraction unaffected was plotted as a function of the log of the dose
to determine the IC50 from the equation log(fa/fu) = mlog(D)  −mlog (IC50).
Dose responses of drugs in combination were tested at a fixed ratio.
Combination index (CI) values were obtained from the equation CIx = Dc1x/D1x +
Dc2x/D2x + Dc1xDc2x/D1xD2x, where Dc1x is dose of drug 1 in combination
required for achieving x percent of cell inviability. Combination index values below 1 are
indication of synergism, near 1 of additivity, and greater than 1 of
antagonism. MTT and NF-κB luciferase
reporter experiments using the drugs as single agents and in combination were
done in triplicates, and all experiments to determine synergism were repeated
at least once. Annexin V-FITC apoptosis assays and western blotting assays were
likewise repeated once. Statistical analyses on replicates were performed by calculating 95% confidence
intervals.


## 3. Results

### 3.1. 17-AAG Synergizes with MS-275 against Synovial Sarcoma In Vitro

To determine if the combination of
Hsp90 inhibitors and HDAC inhibitors are able to synergize on synovial sarcoma,
an in vitro MTT cell
proliferation assay was performed on the synovial sarcoma cell lines SYO-1 and
Fuji using the Hsp90 inhibitor 17-AAG and the HDAC inhibitor MS-275. Cells were
grown in monolayer culture and exposed to varying concentrations of each agent
alone to determine IC50 values. Both agents are effective at
reducing cell proliferation on both synovial sarcoma cell lines in a dose- and
time-dependent manner, with IC50 values shown in Table 1 for SYO-1.
Both are more effective at inhibiting the growth of the synovial sarcoma cell
line SYO-1 than equimolar doxorubicin. Whereas 1 μM of doxorubicin reduces the number of viable SYO-1
cells by 7%, 1 μM
of 17-AAG and 1 μM
of MS-275 reduce the number of cells by 42% and 11%, respectively. Based on
these results, the cell lines were then treated in combinations at multiples of
a set ratio of 2 parts of
17-AAG to 5 parts of
MS-275 and tested for cell proliferation. 17-AAG and MS-275 in combination
showed much greater effectiveness at lower doses for reducing cell
proliferation on both synovial sarcoma cell lines, in a dose- and time-dependent
manner (Figure 1), than either agent alone, with combination index values below
1, indicating synergy.Table 1
Comparison of IC50 values at 24- and 48 hour
Timepoints for MS-275 and 17-AAG as single agents and in combination using MTT
assay.
Timepoint (hrs)IC50 averageMS-27517-AAGCombination (μM)(μM)(μM)MS-27517-AAG244.52.10.60.25480.560.320.30.12Effect of 17-AAG and MS-275 in combination on SYO-1 synovial sarcoma monolayer cultures. Cell survival at  (a) 24 hours and  (b) 48 hours for drugs combined at a set ratio of 2 parts 17-AAG to 5 parts MS-275. MTT cell proliferation assays were performed for the indicated doses, and survival is plotted relative to vehicle control. Corresponding combination indices are shown for  (c)  24 and (d)  48 hours, where values below 1 indicate synergistic drug interactions. Error bars represent 95% confidence intervals. Combination indices were calculated using the Chou and Talalay Median Dose Method (not applicable at low doses where effective cell killing relative to control cell death was not observed).
(a)(b)(c)(d)To confirm synergy, the Annexin V-FITC
apoptosis assay was also used at 24 hours and 48 hours. Efficacy of 17-AAG
and MS-275 as single agents on synovial
sarcoma was confirmed by these assays at all time points in a time- and dose-dependent
manner (data not shown). In combination at a set ratio of 2 parts of 17-AAG to 5 parts of MS-275, greater
apoptosis was observed at lower doses, again indicating drug synergy. Combination
index values were as low as 0.11 and 0.07 for 24 hours and 48 hours,
respectively.


### 3.2. Mechanism of Synergy Involves 17-AAG Abrogation of MS-275-Induced NF-κB Activation

The IκBα complex acts as an NF-κB inhibitor by binding to NF-κB dimers, shuttling them to the cytoplasm, and
retaining them there. IκBα levels are inversely proportional to NF-κB activation. As an inhibitor of the NF-κB pathway, it has been found that 17-AAG is
capable of maintaining levels of IκBα [40]. To explore the possibility of synergy
through a mechanism involving NF-κB, protein
levels of IκBα were measured in response to 17-AAG and MS-275
individually and in combination with synovial sarcoma cells. By western blot, IκBα levels
decrease with MS-275 treatment in a dose-dependent manner, indicating
activation of the NF-κB pathway;
an effect which is abrogated by adding 17-AAG (Figure 2).Dose Response of IκBα. Immuunoblots and quantitation of IκBα levels after treatment with  (a) MS-275,  (b) 17-AAG and  (c)  Combination. Following 24 hour treatment total lysates were prepared and quantified. 10 μg of lysate was run on an SDS-page gel. By immunoblotting, IκBα and p85 (as a loading control) were detected using α-IκBα and α-p85 antibodies. Protein levels following treatment are compared to vehicle control which is set at 1.00.
(a)(b)(c)NF-κB
activation requires relocation of NF-κB
heterodimers from the cytoplasm to the nucleus before it can mediate its
transcriptional effects on cell survival. In synovial sarcoma cells, we find
that nuclear levels of the RelA subunit of NF-κB increase during MS-275 treatment, but
decrease in the presence of 17-AAG as a single agent or in combination with
MS-275 (Figure 3).Dose Response of RelA. Immunoblots and quantitation of nuclear RelA after treatment with (a)  MS-275,  (b ) 17-AAG and (c)  Combination. Following 24 hour treatment, nuclear and cytoplasmic extracts were prepared and quantified. 15 μg were run on an SDS-page gel. Using immunoblotting techiniques, RelA and p85 (as a loading control) were detected using α-RelA and α-p85 antibodies. Protein levels following treatment are compared to vehicle control which is set at 1.00.
(a)(b)(c)Finally, we looked
at the transcriptional potential of NF-κB
following treatment with these drugs using an NF-κB luciferase reporter (Figure 4). MS-275
dramatically increases NF-κB
transcriptional activity (almost 20-fold at higher doses), whereas 17-AAG as a
single agent decreases transcriptional activity. In the combination, the 17-AAG
effect predominates, and there is a net inhibition of NF-κB transcriptional activity. Similar results
were found with Fuji synovial sarcoma cells (data not shown).Transcriptional activation of NF-κB luciferase reporter. NF-κB induction following treatment with  (a)  MS-275, (b)  17-AAG, and (c)  Combination. SYO-1 cells were grown as monolayer cultures in 24 well plates and transfected with 0.3 μg of NF-κB luciferase reporter plasmid. Cells were
treated the following day for 24 hours and lysed with passive lysis buffer. Samples were aliquoted to plates and simultaneously assayed for luminosity by injection with 50 μl/well of LARII reagent and protein quantity by copper sulfate/bichionic acid assay. Readings for luminosity were normalized to protein concentration and vehicle control was set to 1.00. Error bars represent 95% confidence intervals for three replicate measurements.
(a)(b)(c)


### 3.3. Histone Deacetylase 3 (HDAC3) siRNA Knockdown Has Similar Effects to MS-275

The RelA subunit of NF-κB
is acetylated by HDAC3 [22], reducing its ability to interact with its
inhibitor IκBα. All HDAC inhibitors previously shown to
kill synovial sarcoma cells [16–18] include HDAC3 among their specific
targets. Therefore, we hypothesized that our observations on MS-275 (killing
synovial sarcoma cells, but activating NF-κB in a manner which can be blocked with 17-AAG)
might be explained specifically by HDAC3 inhibition. We find that siRNA
directed against HDAC3 kills
SYO-1 cells, whereas the scrambled siRNA control does not (Figure 5(a)).
Furthermore, HDAC3 knockdown
induces RelA nuclear translocation, and this effect is largely abrogated by
adding 17-AAG (Figure 5(b)).
Thus, the same effects on cell survival and NF-κB activation are seen with HDAC3 siRNA knockdown as with the
HDAC inhibitor drug MS-275.HDAC3 siRNA knockdown recapitulates the effects HDAC inhibitors on synovial sarcoma cells.  (a)  50 nM HDAC3 siRNA promotes extensive cell death at 24 hours, as shown by staining of a large inviable cell fraction by 500 ng/mL propidium iodide, where virtually all cells exposed to equimolar scrambled siRNA control are viable. (b) Densitometric quantitation of Western blots for nuclear RelA, expressed relative to control siRNA transfected cells treated with vehicle (1.0).
(a)(b)


### 3.4. MS-275 Synergizes with NF-κB Inhibitors

The compound
BAY 11-7085 is a commercially available inhibitor of NF-κB [41]. In our NF-κB luciferase reporter assay system, BAY 11-7085
strongly represses NF-κB activity
in SYO-1 cells with an IC50 of 4.9 μM at 24 hours. The combination of BAY 11-7085 and MS-275 showed combination index values
as low as 0.26 for 24 hours and 0.25 for 48 hours, indicating
synergism between this NF-κB
inhibitor and the HDAC inhibitor MS-275 (Table 2).Table 2
Combination of HDAC and
NF-κB inhibitors against synovial sarcoma cells.
Timepoint (hrs)IC50 averageMS-275BAY 11-7085Combination (μM)(μM)(μM)MS-275BAY 11244.53.50.451.4480.563.40.270.8


## 3.1. 17-AAG Synergizes with MS-275 against Synovial Sarcoma In Vitro

To determine if the combination of
Hsp90 inhibitors and HDAC inhibitors are able to synergize on synovial sarcoma,
an in vitro MTT cell
proliferation assay was performed on the synovial sarcoma cell lines SYO-1 and
Fuji using the Hsp90 inhibitor 17-AAG and the HDAC inhibitor MS-275. Cells were
grown in monolayer culture and exposed to varying concentrations of each agent
alone to determine IC50 values. Both agents are effective at
reducing cell proliferation on both synovial sarcoma cell lines in a dose- and
time-dependent manner, with IC50 values shown in Table 1 for SYO-1.
Both are more effective at inhibiting the growth of the synovial sarcoma cell
line SYO-1 than equimolar doxorubicin. Whereas 1 μM of doxorubicin reduces the number of viable SYO-1
cells by 7%, 1 μM
of 17-AAG and 1 μM
of MS-275 reduce the number of cells by 42% and 11%, respectively. Based on
these results, the cell lines were then treated in combinations at multiples of
a set ratio of 2 parts of
17-AAG to 5 parts of
MS-275 and tested for cell proliferation. 17-AAG and MS-275 in combination
showed much greater effectiveness at lower doses for reducing cell
proliferation on both synovial sarcoma cell lines, in a dose- and time-dependent
manner (Figure 1), than either agent alone, with combination index values below
1, indicating synergy.Table 1
Comparison of IC50 values at 24- and 48 hour
Timepoints for MS-275 and 17-AAG as single agents and in combination using MTT
assay.
Timepoint (hrs)IC50 averageMS-27517-AAGCombination (μM)(μM)(μM)MS-27517-AAG244.52.10.60.25480.560.320.30.12Effect of 17-AAG and MS-275 in combination on SYO-1 synovial sarcoma monolayer cultures. Cell survival at  (a) 24 hours and  (b) 48 hours for drugs combined at a set ratio of 2 parts 17-AAG to 5 parts MS-275. MTT cell proliferation assays were performed for the indicated doses, and survival is plotted relative to vehicle control. Corresponding combination indices are shown for  (c)  24 and (d)  48 hours, where values below 1 indicate synergistic drug interactions. Error bars represent 95% confidence intervals. Combination indices were calculated using the Chou and Talalay Median Dose Method (not applicable at low doses where effective cell killing relative to control cell death was not observed).
(a)(b)(c)(d)To confirm synergy, the Annexin V-FITC
apoptosis assay was also used at 24 hours and 48 hours. Efficacy of 17-AAG
and MS-275 as single agents on synovial
sarcoma was confirmed by these assays at all time points in a time- and dose-dependent
manner (data not shown). In combination at a set ratio of 2 parts of 17-AAG to 5 parts of MS-275, greater
apoptosis was observed at lower doses, again indicating drug synergy. Combination
index values were as low as 0.11 and 0.07 for 24 hours and 48 hours,
respectively.


## 3.2. Mechanism of Synergy Involves 17-AAG Abrogation of MS-275-Induced NF-κB Activation

The IκBα complex acts as an NF-κB inhibitor by binding to NF-κB dimers, shuttling them to the cytoplasm, and
retaining them there. IκBα levels are inversely proportional to NF-κB activation. As an inhibitor of the NF-κB pathway, it has been found that 17-AAG is
capable of maintaining levels of IκBα [40]. To explore the possibility of synergy
through a mechanism involving NF-κB, protein
levels of IκBα were measured in response to 17-AAG and MS-275
individually and in combination with synovial sarcoma cells. By western blot, IκBα levels
decrease with MS-275 treatment in a dose-dependent manner, indicating
activation of the NF-κB pathway;
an effect which is abrogated by adding 17-AAG (Figure 2).Dose Response of IκBα. Immuunoblots and quantitation of IκBα levels after treatment with  (a) MS-275,  (b) 17-AAG and  (c)  Combination. Following 24 hour treatment total lysates were prepared and quantified. 10 μg of lysate was run on an SDS-page gel. By immunoblotting, IκBα and p85 (as a loading control) were detected using α-IκBα and α-p85 antibodies. Protein levels following treatment are compared to vehicle control which is set at 1.00.
(a)(b)(c)NF-κB
activation requires relocation of NF-κB
heterodimers from the cytoplasm to the nucleus before it can mediate its
transcriptional effects on cell survival. In synovial sarcoma cells, we find
that nuclear levels of the RelA subunit of NF-κB increase during MS-275 treatment, but
decrease in the presence of 17-AAG as a single agent or in combination with
MS-275 (Figure 3).Dose Response of RelA. Immunoblots and quantitation of nuclear RelA after treatment with (a)  MS-275,  (b ) 17-AAG and (c)  Combination. Following 24 hour treatment, nuclear and cytoplasmic extracts were prepared and quantified. 15 μg were run on an SDS-page gel. Using immunoblotting techiniques, RelA and p85 (as a loading control) were detected using α-RelA and α-p85 antibodies. Protein levels following treatment are compared to vehicle control which is set at 1.00.
(a)(b)(c)Finally, we looked
at the transcriptional potential of NF-κB
following treatment with these drugs using an NF-κB luciferase reporter (Figure 4). MS-275
dramatically increases NF-κB
transcriptional activity (almost 20-fold at higher doses), whereas 17-AAG as a
single agent decreases transcriptional activity. In the combination, the 17-AAG
effect predominates, and there is a net inhibition of NF-κB transcriptional activity. Similar results
were found with Fuji synovial sarcoma cells (data not shown).Transcriptional activation of NF-κB luciferase reporter. NF-κB induction following treatment with  (a)  MS-275, (b)  17-AAG, and (c)  Combination. SYO-1 cells were grown as monolayer cultures in 24 well plates and transfected with 0.3 μg of NF-κB luciferase reporter plasmid. Cells were
treated the following day for 24 hours and lysed with passive lysis buffer. Samples were aliquoted to plates and simultaneously assayed for luminosity by injection with 50 μl/well of LARII reagent and protein quantity by copper sulfate/bichionic acid assay. Readings for luminosity were normalized to protein concentration and vehicle control was set to 1.00. Error bars represent 95% confidence intervals for three replicate measurements.
(a)(b)(c)


## 3.3. Histone Deacetylase 3 (HDAC3) siRNA Knockdown Has Similar Effects to MS-275

The RelA subunit of NF-κB
is acetylated by HDAC3 [22], reducing its ability to interact with its
inhibitor IκBα. All HDAC inhibitors previously shown to
kill synovial sarcoma cells [16–18] include HDAC3 among their specific
targets. Therefore, we hypothesized that our observations on MS-275 (killing
synovial sarcoma cells, but activating NF-κB in a manner which can be blocked with 17-AAG)
might be explained specifically by HDAC3 inhibition. We find that siRNA
directed against HDAC3 kills
SYO-1 cells, whereas the scrambled siRNA control does not (Figure 5(a)).
Furthermore, HDAC3 knockdown
induces RelA nuclear translocation, and this effect is largely abrogated by
adding 17-AAG (Figure 5(b)).
Thus, the same effects on cell survival and NF-κB activation are seen with HDAC3 siRNA knockdown as with the
HDAC inhibitor drug MS-275.HDAC3 siRNA knockdown recapitulates the effects HDAC inhibitors on synovial sarcoma cells.  (a)  50 nM HDAC3 siRNA promotes extensive cell death at 24 hours, as shown by staining of a large inviable cell fraction by 500 ng/mL propidium iodide, where virtually all cells exposed to equimolar scrambled siRNA control are viable. (b) Densitometric quantitation of Western blots for nuclear RelA, expressed relative to control siRNA transfected cells treated with vehicle (1.0).
(a)(b)


## 3.4. MS-275 Synergizes with NF-κB Inhibitors

The compound
BAY 11-7085 is a commercially available inhibitor of NF-κB [41]. In our NF-κB luciferase reporter assay system, BAY 11-7085
strongly represses NF-κB activity
in SYO-1 cells with an IC50 of 4.9 μM at 24 hours. The combination of BAY 11-7085 and MS-275 showed combination index values
as low as 0.26 for 24 hours and 0.25 for 48 hours, indicating
synergism between this NF-κB
inhibitor and the HDAC inhibitor MS-275 (Table 2).Table 2
Combination of HDAC and
NF-κB inhibitors against synovial sarcoma cells.
Timepoint (hrs)IC50 averageMS-275BAY 11-7085Combination (μM)(μM)(μM)MS-275BAY 11244.53.50.451.4480.563.40.270.8


## 4. Discussion

In current clinical practice, available systemic therapies
for synovial sarcoma have limited effectiveness and have not been definitively
proven to increase cure rates. Recent research building on gene expression
profiling data has identified several promising agents active against synovial
sarcoma [42]. 17-AAG is effective against synovial sarcoma preclinical models,
but clinical trials in other tumors have shown some toxicity at higher doses including evidence
of liver toxicity, optic neuritis, dyspnea, fatigue, nausea, vomiting,
anorexia, diarrhea, anemia, and low grade fever [43, 44]. Similarly, in high
doses, MS-275 has shown such toxicity
as nausea, vomiting, anorexia, fatigue, hypoalbuminemia, and hypocalcemia in
clinical trials [45].The HDAC inhibitor romidepsin has additionally been associated
with atrial fibrillation, tachycardia, and in one case a cardiac sudden death [46].
Synergism would allow lower doses to be used. While synergism has been
demonstrated by others between 17-AAG and HDAC inhibitors in myelogenous
leukemia cells [10, 28], our study is the first to demonstrate that the orally
available agent MS-275 synergizes with 17-AAG. This is also the first
demonstration of synergism between Hsp90 and HDAC inhibitors in synovial
sarcoma; an often fatal disease wherein each of these drug classes has recently
been demonstrated to show activity in
vitro [6, 16]. Here we have shown that combinations of the Hsp90
inhibitor 17-AAG and the HDAC inhibitor MS-275 are synergistic, requiring as
little as ninefold less drug to achieve equivalent effects in vitro against synovial sarcoma
models.Our results
provide evidence that synergism between 17-AAG and MS-275 might be mediated by
the prosurvival NF-κB pathway.
Evidence from microarray expression profiling has demonstrated that the NF-κB activator RIPK4 is highly upregulated in synovial sarcoma, and the data
presented here shows that the RelA subunit of NF-κB is found in the nucleus of untreated synovial
sarcoma cells, suggesting baseline activation of NF-κB in this malignancy. In addition, the TLE
transcriptional corepressor, which is expressed at extremely high levels in
synovial sarcoma cells [47], forms a complex with HDACs at NF-κB target sites [48]. Consistent with a role for
NF-κB in
synovial sarcoma growth, we show that a chemical inhibitor of NF-κB (BAY 11-7085) has in vitro activity against synovial sarcoma cells as a single
agent. Although additional mechanisms may contribute to the observed synergy,
in separate experiments, we found that neither 17-AAG nor HDAC inhibitors
altered the level of SYT-SSX protein expression, nor are Hsp90 levels or acetylation status altered by HDAC inhibitor
treatment (data not shown).In conclusion, the Hsp90 inhibitor 17-AAG and the HDAC
inhibitor MS-275 have synergistic, antiproliferative,
and proapoptotic effects on synovial sarcoma in vitro. 17-AAG and MS-275 in combination reverse the
activation of NF-κB seen
with MS-275 alone, as measured by levels of the NF-κB inhibitory IκBα complex.
Net effects of these drugs on nuclear levels of the active NF-κB subunit RelA and on NF-κB luciferase reporter transcription are
consistent with these findings. The observed effects of MS-275 on NF-κB can be recapitulated by knocking down HDAC3; an enzyme which includes RelA
as one of its nonhistone substrates and which is a target of MS-275,
depsipeptide, and several other HDAC inhibitors. In addition, the NF-κB inhibitor BAY 11-7085 is also synergistic
with MS-275. Agents inhibiting NF-κB in
combination with the HDAC inhibitor MS-275 show promising in vitro activity against synovial
sarcoma; an often-fatal disease of young adults for which development of truly effective
systemic therapies is needed.



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*Source: 794901-2009-03-24.xml*