From www.bloodjournal.org by on November 22, 2010. For personal use only.

2008 112: 1886-1893

Prepublished online Jun 30, 2008;
doi:10.1182/blood-2008-03-143644

HDAC6 inhibition enhances 17-AAGmediated abrogation of hsp90

chaperone function in human leukemia cells
Rekha Rao, Warren Fiskus, Yonghua Yang, Pearl Lee, Rajeshree Joshi, Pravina Fernandez, Aditya
Mandawat, Peter Atadja, James E. Bradner and Kapil Bhalla

Updated information and services can be found at:

http://bloodjournal.hematologylibrary.org/cgi/content/full/112/5/1886
Articles on similar topics may be found in the following Blood collections:
Neoplasia (4217 articles)

Information about reproducing this article in parts or in its entirety may be found online at:
http://bloodjournal.hematologylibrary.org/misc/rights.dtl#repub_requests

Information about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/misc/rights.dtl#reprints

Information about subscriptions and ASH membership may be found online at:
http://bloodjournal.hematologylibrary.org/subscriptions/index.dtl

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published

semimonthly by the American Society of Hematology, 1900 M St, NW, Suite
200, Washington DC 20036.
Copyright 2007 by The American Society of Hematology; all rights reserved.
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
NEOPLASIA

HDAC6 inhibition enhances 17-AAG–mediated abrogation of hsp90 chaperone

function in human leukemia cells
Rekha Rao,1 Warren Fiskus,1 Yonghua Yang,1 Pearl Lee,1 Rajeshree Joshi,1 Pravina Fernandez,1 Aditya Mandawat,1
Peter Atadja,2 James E. Bradner,3 and Kapil Bhalla1
1Medical College of Georgia (MCG) Cancer Center, Augusta; 2Novartis Institute for Biomedical Research, Cambridge, MA; and 3Dana-Farber Cancer Institute,

Boston, MA

Histone deacetylase 6 (HDAC6) is a heat
shock protein 90 (hsp90) deacetylase.
Treatment with pan-HDAC inhibitors or
depletion of HDAC6 by siRNA induces
hyperacetylation and inhibits ATP bind-
ing and chaperone function of hsp90.
Treatment with 17-allylamino-demothoxy
geldanamycin (17-AAG) also inhibits ATP
binding and chaperone function of hsp90,
resulting in polyubiquitylation and protea-
somal degradation of hsp90 client pro-
teins. In this study, we determined the
effect of hsp90 hyperacetylation on the
anti-hsp90 and antileukemia activity of
17-AAG. Hyperacetylation of hsp90 in-
creased its binding to 17-AAG, as well as
enhanced 17-AAG–mediated attenuation
of ATP and the cochaperone p23 binding
to hsp90. Notably, treatment with 17-AAG
alone also reduced HDAC6 binding to
hsp90 and induced hyperacetylation of
hsp90. This promoted the proteasomal
degradation of HDAC6. Cotreatment with
17-AAG and siRNA to HDAC6 induced
more inhibition of hsp90 chaperone func-
tion and depletion of BCR-ABL and c-Raf
than treatment with either agent alone. In
addition, cotreatment with 17-AAG and
tubacin augmented the loss of survival of
K562 cells and viability of primary acute
myeloid leukemia (AML) and chronic my-
eloid leukemia (CML) samples. These find-
ings demonstrate that HDAC6 is an hsp90
client protein and hyperacetylation of
hsp90 augments the anti-hsp90 and anti-
leukemia effects of 17-AAG. (Blood. 2008;
112:1886-1893)

Introduction
Among the stress-inducible molecular chaperones, hsp90 is an
abundantly expressed (as much as 2% of total cellular protein),
homodimeric, ATP-dependent protein.1-3 Hsp90 is required for the
maintenance of native and functionally active conformation of
important signaling protein kinases and transcription factors, also
known as hsp90 client proteins.1-3 Hsp90-based chaperone com-
plex interacts with its client proteins in an iterative manner, in
which hsp90 cycles between ATP- or ADP-bound conformations
mediated through multiple rounds of ATP binding and hydroly-
sis.4,5 Replacement of ADP by ATP in hsp90 alters hsp90 conforma-
tion, thereby releasing cochaperones p60HOP and hsp70/hsp40
complex, while simultaneously recruiting another set of cochaper-
ones including p23 and cyclophillin 40 (when the client protein is a
steroid nuclear hormone receptor) or p50cdc37 (when the client
protein is a signaling protein kinase).2,3,6-9 Conversely, ATP hydro-
lysis due to its intrinsic ATPase activity creates the ADP-bound
conformation of hsp90, which directs the misfolded client protein
to a covalent linkage with polyubiquitin and subsequent degrada-
tion by the 26S proteasome.2,3
Benzoquinone ansamycin–derived antibiotic geldanamycin (GA)
and its analogs (eg, 17-AAG and 17-DMAG) bind to the ATP/ADP-
binding pocket with higher affinity than the nucleotide, replacing
the nucleotide and inhibiting the chaperone function of hsp90.2,3 By
blocking ATP binding, treatment with 17-AAG stabilizes ADP-
bound hsp90 into a conformation that recruits hsp70-based cochap-
erone complex, which allows polyubiquitylation of the misfolded
client protein by specific E3 ubiquitin ligase and directs the client
proteins to proteasomal degradation.2,3 A growing number of hsp90
client proteins have been recognized in human acute leukemia
cells.2,3 These include conformationally metastable signaling pro-
tein kinases and mutated oncoprotein kinases (eg, FLT-3, Bcr-Abl,
NPM-ALK, c-Raf, AKT, and CDK4).1-3,10-14 Treatment with 17-
AAG has been shown to deplete hsp90 client proteins, which are
essential for growth and survival of transformed cells. Conse-
quently, 17-AAG exerts lethal in vitro and in vivo effects against
human leukemia cells.10,15 Recently, posttranslational modifica-
tions such as hyperphosphorylation, S-nitrosylation, and reversible
hyperacetylation of lysine residues have been demonstrated to
affect cochaperone association, ATP binding, and chaperone func-
tion of hsp90.2 The predominantly cytosolic, class IIB HDAC
family member HDAC6 was shown to be the deacetylase for
hsp90, ␣-tubulin, and cortactin.16-18 Depletion of HDAC6 levels, or
the inhibition of its deacetylase activity with pan-histone deacety-
lase inhibitors (HDIs), was shown to result in reversible hyperacety-
lation of hsp90.16,19 This was associated with inhibition of ATP and
client protein binding to hsp90, inhibition of hsp90 chaperone
function and proteasomal degradation of hsp90 client proteins.16,19
It is noteworthy that since the mutant forms of oncoprotein kinases
(eg, Bcr-Abl and FLT-3) may be more dependent on the chaperone
function of hsp90, inhibition of hsp90 may be particularly effective
in depleting the levels of the mutant Bcr-Abl and FLT-3.16,19,20
Furthermore, we have previously demonstrated that cotreatment
with 17-AAG and HDI exerts synergistic cytotoxic effects against
Bcr-Abl– and FLT-3–expressing human leukemia cells.13 It is
likely that there are multiple possible mechanisms underlying the
synergistic antileukemia effects of the combination of HDI and

Submitted March 4, 2008; accepted June 9, 2008. Prepublished online as payment. Therefore, and solely to indicate this fact, this article is hereby
Blood First Edition paper, June 30, 2008; DOI 10.1182/blood-2008-03-143644. marked ‘‘advertisement’’ in accordance with 18 USC section 1734.

The publication costs of this article were defrayed in part by page charge © 2008 by The American Society of Hematology

1886 BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

 From www.bloodjournal.org by on November 22, 2010. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
17-AAG. However, in the present study, we focused on how hsp90
hyperacetylation influences 17-AAG–mediated inhibition of hsp90.
Specifically, we have determined that induction of hsp90 hyperacety-
lation (1) enhances the binding of 17-AAG to hsp90, (2) augments
17-AAG–mediated inhibition of ATP and cochaperone binding to
hsp90, and (3) promotes 17-AAG–mediated depletion of hsp90
client proteins. Our findings also show that HDAC6 is an hsp90
client protein, and treatment with 17-AAG promotes the degrada-
tion of HDAC6 by the proteasome.
Methods
Informed consent was obtained in accordance with the Declaration of
Helsinki. The study was approved by the Medical College of Georgia’s
Human Assurance Committee (IRB).
Reagents and antibodies
17-AAG was obtained from Developmental Therapeutics Branch of the
Cancer Treatment Evaluation Program, National Cancer Institute/National
Institutes of Health (NIH; Bethesda, MD). LBH589 and LAQ824 were
provided by Novartis Pharmaceuticals (East Hanover, NJ). ATP-Sepharose
was purchased from Innova Biosciences (Cambridge, United Kingdom).
Anti-hsp90 and -hsp70 antibodies were purchased from StressGen Biotech-
nologies (Victoria, BC). Monoclonal anti–acetyl lysine antibody was
purchased from Cell Signaling Technology (Beverly, MA). Monoclonal
anti–acetyl ␣-tubulin was purchased from Sigma-Aldrich (St Louis, MO).
Monoclonal anti-Abl and HDAC6 antibodies were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell culture
Human chronic myeloid leukemia (CML) K562 cells, acute myeloid
leukemia (AML) HL-60 cells, and the bone marrow stroma HS-5 cells were
maintained as previously described.16
Western blot analyses and immunoprecipitation
Western blot analyses of Bcr-Abl, c-Raf-1, AKT, hsp90, hsp70, HDAC6,
and ␤-actin were performed using specific antisera or monoclonal antibod-
ies (listed in “Regents and antibodies”), as described previously.16,19-21
Bcr-Abl and hsp90 were immunoprecipitated as previously described.16,19
The expression of ␤-actin was used as a loading control.
Acetylation of Hsp90 and its binding to ATP-Sepharose
For hsp90 ATP-binding studies, cell lysates were prepared after drug
treatment and hsp90 was affinity precipitated from 200 ␮g total cellular
protein using ATP-Sepharose beads. The hsp90 in the precipitates was
assessed by Western blot analysis using a monoclonal anti-hsp90 antibody.
For hsp90 acetylation, immunoprecipitated hsp90 was immunoblotted and
acetylated hsp90 was detected using an anti–acetyl lysine antibody.16
Preparation of detergent-soluble and -insoluble fractions
After the designated drug treatments, NP-40–soluble and –insoluble
fractions were prepared as described previously.16,22 Proteins from the
NP-40 soluble and insoluble fractions (50 ␮g) were separated on 7.5%
SDS–polyacrylamide gel and analyzed by Western blotting.16,19
Transfection of HDAC6 siRNA vector
K562 cells were transiently transfected as per the manufacturer’s instruc-
tions, using Amaxa Nucleofector Electroporator (Gaithersburg, MD) with
the plasmid vector pBS/U6 with or without the HDAC6 siRNA,16 which
had a 21-nucleotide sequence of 5⬘-GG ATG GAT CTG AAC CTT GAG A-
3⬘, corresponding to the targeted nucleotide sequence 200-219 in the
HDAC6 mRNA (accession no. BC013737).23
HYPERACETYLATION OF hsp90 AND 17-AAG ACTIVITY 1887
Biotinylated-GM–binding assay
Biotinylated-GM binding to hsp90 was assessed as described previ-
ously.22 Briefly, 48 hours after transfection, control- and siHDAC6-
transfected cell lysates were incubated with increasing concentration of
17-AAG, and biotinylated-GM (B-GM) was added as a competitor to
displace 17-AAG from hsp90. This was followed by immunoprecipita-
tion of biotinylated-GM–bound hsp90 using Streptavidin-agarose beads
and immunoblotting for hsp90.
Confocal immunofluorescence microscopy to evaluate Ki-67
expression
Following vector or siHDAC6 transfection for 48 hours, cells were fixed
with 4% paraformaldehyde for 10 minutes. Following this, the slides were
blocked with 3% BSA for 30 minutes and incubated with anti-HDAC6 and
anti–Ki-67 antibody.24 After 3 washes with PBS, the slides were incubated
in Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Molecular
Probes, Invitrogen, Eugene, OR) for 1 hour at 1:3000 dilution. After
3 washes with PBS, the cells were counterstained with DAPI using
Vectashield mountant with DAPI and imaged using Zeiss LSM510 confocal
microscope (Carl Zeiss, Heidelberg, Germany), as previously described.25
Colony growth inhibition, loss of viability, and apoptosis
assessment
After vector or siHDAC6 transfection for 48 hours, cells were treated with
the designated concentrations of 17-AAG for 24 hours; untreated and
drug-treated cells were washed in RPMI 1640 medium. After this, 200 cells
treated under each condition were resuspended in 100 ␮L RPMI 1640
media containing 10% FBS, then plated in duplicate wells in a 12-well plate
containing 1.0 mL Methocult media (StemCell Technologies, Vancouver,
BC) per well. The plates were placed in an incubator at 37°C with 5% CO2
for 10 days. After this incubation, colonies consisting of 50 or more cells, in
each well, were counted by an inverted microscope and percentage of
colony growth inhibition compared with the untreated control cells was
calculated.16,21 Viability of cells incubated with the drugs was performed
using trypan blue exclusion assay after 48 hours. The percentage of
apoptotic cells was determined by annexin V/propidium iodide (PI) staining
and/or by assessing the fraction of hypodiploid (sub-G1) cells, using flow
cytometry as previously described.19,20
Statistical analyses
Data were expressed as means plus or minus SEM. Comparisons used
Student t test or ANOVA, as appropriate. Values of P less than .05 were
assigned significance.
Results
HDAC6 inhibition induces hyperacetylation of hsp90 and hsp70
We first compared the effect of the pan-HDAC inhibitor LBH589
and the selective HDAC6 knockdown on hsp90 and hsp70
acetylation in acute leukemia cells. Previous reports had indicated
that LBH589 is a potent pan-HDAC, and especially HDAC6
inhibitor.26 Consistent with these reports, treatment with LBH589,
or knockdown of HDAC6 protein levels using an siRNA to
HDAC6, caused ␣-tubulin acetylation, as well as induced hyper-
acetylation of hsp90 and hsp70 (Figure 1A,B). Similar findings
were also observed after treatment of human leukemia HL-60 cells
with LBH589 (data not shown). Consistent with our previous
report,16 similar to treatment with siRNA to HDAC6, LBH589
treatment also increased the amount of polyubiquitylated proteins
in the immunoprecipitate with anti–Bcr-Abl antibody (Figure 1D),
as well as caused the depletion of Bcr-Abl and c-RAF levels in
K562 cells (Figure 1E).
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
1888 RAO et al BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

A K562
D Control siHDAC6
Control siHDAC6
0 100 0 nM LBH589
0 100 0 nM LBH589
IP: Abl
IB:
HDAC6

acetyl tubulin
Ubiquitin
β actin

B Control siHDAC6
0 100 0 nM LBH589
IP hsp90 Bcr-Abl
IB:
acetyl lysine
hsp90
IP hsp70 E Control siHDAC6
IB:
acetyl lysine
0 100 0 nM LBH589
hsp70 IB:
Bcr-Abl

C
K562 HL-60 c-Raf
0 100 250 0 100 250 nM, LAQ824
IP: hsp90 β-actin
IB:
acetyl lysine
hsp90

Figure 1. Hydroxamic acid analogues induce acetylation of hsp90 and hsp70. (A) K562 cells were transfected with control or siHDAC6 constructs. After 48 hours, they
were exposed to the indicated does of LBH589 for 16 hours and immunoblotted for HDAC6, acetyl-tubulin, or ␤-actin. (B) After 48 hours of control vector or siHDAC6
transfection and indicated exposure to LBH589, hsp90 and hsp70 were immunoprecipitated and immunoblotted for acetyl lysine or indicated chaperone/cochaperone.
(C) K562 and HL-60 cells were exposed to the indicated concentrations of LAQ824 for 16 hours. After this, hsp90 was immunoprecipitated from the cell lysates and
immunoblotted with either anti-hsp90 or anti–acetylated lysine antibody. (D) The cell lysates from K562 cells expressing HDAC6 siRNA or from untreated or
LBH589-treated K562 cells were immunoprecipitated with anti-Abl antibody, and the immunoprecipitates were immunoblotted with antiubiquitin. The blot was stripped
and immunoblotted with anti-Abl antibody. (E) The cell lysates were immunoblotted with anti-Abl or anti–c-Raf antibody. The levels of ␤-actin served as the
loading control.

Hyperacetylated hsp90 has a higher affinity for 17-AAG
Given that hsp90 from tumor cells has higher affinity for
17-AAG,22 we determined whether knockdown of HDAC6 affects
the affinity of hsp90 for 17-AAG. Using the biotinylated geldana-
mycin (B-GM)–binding assay, we observed that HDAC6 knock-
down increased the affinity of 17-AAG for hsp90, as evidenced by
the decreased in vitro ability of B-GM to displace 17-AAG from
hsp90 (Figure 2A). We further evaluated whether 17-AAG affects
hsp90 acetylation in vivo. As shown in Figure 2B, treatment with
3.0 ␮M 17-AAG for 16 hours alone induced hsp90 acetylation to
an extent that is comparable with hsp90 acetylation after HDAC6
knockdown. No significant increase in hsp90 acetylation was
detected in cells in which HDAC6 had been knocked down by
treatment with 17-AAG, either because the anti–acetyl lysine
antibody is unable to detect further increase in acetylation or
because the exposure to either condition alone achieved a
saturation threshold for hsp90 acetylation. Lower levels of
hsp90 acetylation were observed with 1.0 ␮M 17-AAG (data not
shown). These observations suggest that 17-AAG induces hyper-
acetylation of hsp90 and hyperacetylated hsp90 has increased
affinity for 17-AAG.
Hyperacetylation of hsp90 increases 17-AAG–mediated
depletion of ATP and p23-binding to hsp90
We next evaluated whether increased binding of 17-AAG to
hyperacetylated hsp90 affects 17-AAG–mediated decline in the
chaperone function of hsp90, which is dependent on its ATP and
cochaperone binding. Control vector or siHDAC6-transfected
cells were treated with 17-AAG for 16 hours and the level of
ATP-bound hsp90 was determined. Our results indicate that the
hyperacetylation of hsp90 mediated by HDAC6 knockdown
depleted ATP binding to hsp90 (Figure 3A). Cotreatment with
17-AAG and siHDAC6 reduced ATP binding to a lower level
than either of the treatments alone (Figure 3A). Knockdown of
HDAC6 slightly increased 17-AAG–mediated induction of the
total hsp90 levels. In addition, hsp90 hyperacetylation was
associated with reduced binding of hsp90 to the cochaperone
p23 but not cdc37 (Figure 3B,C). Reduced binding of p23 to
hyperacetylated hsp90 is consistent with its reduced binding to
ATP. This is consistent with previous observation that p23 binds
only the ATP-bound form of hsp90, which facilitates the
maturation of hsp90 client proteins.4,5
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5 HYPERACETYLATION OF hsp90 AND 17-AAG ACTIVITY 1889

Biotin-GM bound to hsp90 HDAC6 is an hsp90 client protein

A
0 10 50 100 1000 nM, 17-AAG
Having established that 17-AAG induces hsp90 acetylation (Figure
IB: hsp90
2A) and enhances the loss of hsp90 chaperone function after
Control HDAC6 inhibition (Figure 3A,B), we hypothesized that HDAC6
1.0 0.9 0.81 0.54 0.21 binds to and is chaperoned by hsp90, making it a bona fide hsp90
siHDAC6- client protein. Indeed, 17-AAG–mediated abrogation of hsp90
transfected chaperone function was associated with a decline in the levels of
1.0 0.38 0.2 0.11 0.07
HDAC6 in K562 cells, as well as in the primary AML cells (Figure
4A), very similar to the other bona fide hsp90 client proteins. This
was accompanied by increased acetylation of ␣-tubulin in both cell
K562 types. Earlier studies have shown that inhibition hsp90 function
B shifts the chaperone association of client proteins from hsp90 to
Control siHDAC6 hsp70 and results in their proteasomal degradation.1-3 Treatment of
0 3 0 3 IgG Input µM AAG, 16 hrs
K562 cells with 17-AAG for 4 hours reduced the binding of hsp90
IP: hsp90
to HDAC6 and reciprocally increased its association with hsp70
IB: (Figure 4B). 17-AAG–mediated depletion of HDAC6 over 8 hours
acetyl lysine was due to the proteasomal degradation of HDAC6, since its level
1.0 2.42 2.46 2.73 was restored by cotreatment with the proteasome inhibitor bort-
ezomib (Figure 4C). This was seen not only in the total cell lysates
hsp90 of K562 cells, but also in the detergent (NP40)–insoluble or the
–soluble fraction of K562 cells (Figure 4D). Collectively, these
Figure 2. Depletion of HDAC6 increases the affinity of acetylated hsp90 for
17-AAG. (A) K562 cells were transfected with control vector or siRNA to HDAC6.
findings indicate that HDAC6 is an hsp90 client protein.
After 48 hours, the cells were harvested and lysed. The lysates were incubated with
or without indicated doses of 17-AAG for 30 minutes at 4°C, and then incubated with Hyperacetylation of hsp90 increases 17-AAG–mediated
biotin-GM for 1 hour at 4°C. To this, washed Streptavidin-agarose beads were added depletion of Bcr-Abl, AKT, and c-Raf
and incubated overnight at 4°C. The immunoprecipitates were washed and proteins
were eluted with SDS sample loading buffer before the immunoblot analyses with Next, we determined whether hyperacetylation of hsp90 by HDAC6
specific antibody against hsp90. Bands in the Western blots were quantified by
knockdown would increase 17-AAG–mediated abrogation of the
densitometry using ImageQuant5.2 (GE Healthcare, Piscataway, NJ). (B) Vector of
siHDAC6-transfected cells were exposed to indicated dose of 17-AAG after 48 hours chaperone association of hsp90 with Bcr-Abl. Figure 5A demon-
of transfection. Hsp90 acetylation was assessed as described in “Acetylation of strates that hyperacetylation of hsp90 enhanced 17-AAG–induced
Hsp90 and its binding to ATP-Sepharose.” Vertical line has been inserted to indicate a
disruption in the chaperone association of Bcr-Abl with hsp90 in a
repositioned lane from the same gel.
time-dependent manner. This resulted in more depletion of Bcr-Abl

K562
A
Control siHDAC6

0 3 5 0 3 5 μM, 17-AAG , 16 Hours

IP: ATP-Sepharose
hsp90
1.0 0.81 0.32 0.63 0.46 0.18

Control siHDAC6
0 3 5 0 3 5 μM, 17-AAG , 16 Hours

hsp90

β-actin

B K562
K562 C
Control siHDAC6
Control siHDAC6
0 3 5 0 3 5 μM, 17-AAG , 16 Hours
0 3 5 0 3 5 μM, 17-AAG , 16 Hours IP: cdc37
IP:p23 IB:
IB:
hsp90
hsp90
1.0 0.66 0.71 0.25 0.15 0.05
cdc37
p23

Figure 3. Knockdown of HDAC6 expression inhibits binding of ATP to hsp90 and affects the binding of hsp90 to its cochaperone p23. (A) K562 cells expressing
HDAC6 siRNA or K562 vector control cells were treated with the indicated dose of 17-AAG for 16 hours. After this treatment, cell lysates were affinity precipitated with
ATP-Sepharose and immunoblotted with anti-hsp90 antibody. The levels of hsp90 after 17-AAG treatment are as indicated. (B) Vector control K562 and HDAC6 siRNA
knockdown cells were left untreated or treated for 16 hours with 3 and 5 ␮M 17-AAG. Lysates were immunoprecipitated with anti-p23 antibody followed by immunoblotting with
anti-hsp90 and anti-p23 antibody. (C) Alternatively, lysates were immunoprecipitated with anti-cdc37 antibody followed by immunoblotting with anti-hsp90 and anti-cdc37
antibody.
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
1890 RAO et al BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

A B IgG 0 1 3 µM 17-AAG, 4 hrs

K562 Primary AML
IPHDAC6
0 1 3 0 1 µM 17-AAG IB:
hsp90
HDAC6
1.0 0.73 0.37
acetyl tubulin hsp70

β-actin
HDAC6

C D NP40-insoluble NP40-soluble

0 3 0 3 µM 17-AAG, 8 hours 0 3 0 3 0 3 0 3 µM 17-AAG

0 0 100 100 nM Bortezomib 0 0 100 100 nM Bortezomib
0 0 100 100
HDAC6 HDAC6
1.00 0.73 1.13 1.20

β-actin c-Raf

β-actin

Figure 4. HDAC6 has chaperone association with hsp90. (A) K562 cells or primary AML sample were treated with indicated doses of 17-AAG for 16 hours and 24 hours,
respectively. After this, the resulting lysates were probed for HDAC6, acetyl-tubulin, and ␤-actin. (B) HDAC6 was immunoprecipitated from the cells incubated for 4 hours with
indicated doses of 17-AAG and immunoblotted with anti-hsp90, anti-hsp70, or anti-HDAC6 antibody. (C) K562 cells were exposed to indicated concentrations of drugs for
8 hours and the resulting cell lysates were immunoblotted for HDAC6 and ␤-actin. (D) K562 cells were exposed to indicated concentration of drugs for 8 hours and
NP-40–insoluble and –soluble fractions were prepared from the cell lysates. The resulting fractions were immunoblotted with anti-HDAC6 or anti–c-Raf antibodies.

levels due to 17-AAG treatment of K562 cells treated with the caused more depletion of Bcr-Abl, AKT, and c-Raf in those cells in
siRNA to HDAC6, compared with those cells treated with the which HDAC6 had been knocked down and hsp90 was hyperacety-
control siRNA (Figure 5B). Importantly, 17-AAG treatment also lated, compared with the control cells (Figure 5B).
Hyperacetylation of hsp90 increases 17-AAG–induced loss of
A clonogenic survival of K562 cells
Control siHDAC6
0 8 16 0 8 16 Hours, 5 μM, 17-AAG Next, we assessed the effect of HDAC6 depletion and hsp90
IP:Abl hyperacetylation on 17-AAG–mediated loss of clonogenic survival
IB:
of K562 cells. Figure 6A demonstrates that knockdown of HDAC6
hsp90
alone markedly inhibited colony growth of K562 cells. Treatment
Bcr-Abl with 17-AAG alone also inhibited the clonogenic survival of K562
cells. In addition, treatment with the siRNA to HDAC6 signifi-
cantly sensitized K562 cells to 17-AAG–mediated loss of clono-
genic survival (P ⬍ .05). Knockdown of HDAC6 by treatment
B with siRNA to HDAC6 also depleted Ki-67 expression in K562
Control siHDAC6
cells, as determined by immunofluorescent confocal microscopy
0 8 16 0 8 16 Hours, 5 μM, 17-AAG
(Figure 6B). This was not associated with induction of apoptosis
Bcr-Abl (sub-G1 fraction or annexin V staining) or with alteration in the
1.00 0.81 0.49 0.93 0.47 0.21 percentage of cells in the S phase of the cell cycle (as determined
AKT by propidium iodide staining and flow cytometry; data not shown).
1.00 0.56 0.43 0.65 0.60 0.20 Cotreatment of K562 cells with 17-AAG and tubacin, an HDAC6-
c-Raf specific inhibitor,27,28 also significantly enhanced the sensitivity of
1.00 0.46 0.05 0.58 0.01 0.00 K562 cells to 17-AAG–mediated loss of viability (Figure 6C). We
also determined the effects of treatment with tubacin and/or
β-actin 17-AAG on 3 primary samples each procured from patients with
relapsed imatinib refractory CML and relapsed AML, as well as
Figure 5. Diminishing the levels of HDAC6 in leukemic cells not only reduces
the binding of hsp90 to its client protein (Bcr-Abl) but also causes partial
CD34⫹ normal bone marrow progenitor cells. Table 1 shows that
depletion of the client protein levels. (A) K562 cells were transfected with vector cotreatment with tubacin and 17-AAG caused more loss of cell
control or HDAC6 siRNA for 48 hours. After this, the cells were treated with 5 ␮M viability than treatment with either agent alone in each of the CML
17-AAG for 0, 8, and 16 hours and cell lysates were immunoprecipitated with anti-Abl
or AML samples tested. Furthermore, the superior effect of the
antibody, and the immunoprecipitates were either immunoblotted with anti-hsp90 or
anti-Abl antibody. (B) Alternatively, the cell lysates were immunoblotted with anti-Abl, combination of the 2 drugs was also observed when 2 CML and
AKT, or anti–c-Raf antibody. The levels of ␤-actin served as the loading control. AML samples each were grown on the human bone marrow–
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5 HYPERACETYLATION OF hsp90 AND 17-AAG ACTIVITY 1891

A derived HS-5 stromal cells (Table 1). These findings suggest that
120
p< 0.001 treatment with tubacin and/or 17-AAG may be able to partially
100
overcome the protective mechanism(s) mediated by the bone
control
marrow stroma. Table 1 also shows that the combination of tubacin
siHDAC6
% colony grow th inhibition

80 and 17-AAG exerts less toxicity against the normal CD34⫹

p<0.05 progenitor cells.
60
p<0.05

40

20 Discussion
0
In this study, we demonstrate for the first time that hyperacetylation
control 1 µM 17-AAG 3 µM 17-AAG
of hsp90 induced either by HDAC6 inhibition after LBH589
treatment, or by the depletion of HDAC6 levels by treatment with
B K562
siRNA to HDAC6, preferentially increased the binding of hsp90 to
17-AAG. Previous studies had demonstrated that inducing hyper-
HDAC6 Ki-67 DAPI MERGE acetylation of hsp90 alone was associated with decreased binding
of hsp90 to ATP, with disruption of the chaperone association of
control hsp90 with GR, AR, and ER␣,18,29,30 as well as with the other
oncoprotein kinases Bcr-Abl, FLT-3, AKT, c-Raf, and HER-
2.16,20,31 Present studies extend these observations by demonstrat-
ing that hyperacetylation of hsp90 enables 17-AAG to more
siHDAC6 potently inhibit hsp90 binding to p23, ATP, and the client proteins.
This augments 17-AAG–mediated inhibition of hsp90 chaperone
function, resulting in depletion of hsp90 client proteins.
Both LBH589 treatment and HDAC6 knockdown decreased
hsp90 binding to ATP and the cochaperone p23. This finding is
C 70
p<0.05
consistent with the notion that p23 binds ATP-bound hsp90 and
K562 locks it in an ATP-dependent conformational state that has high
60 p<0.001
affinity for client proteins.32 In contrast, the binding of hsp90 to
50 cdc37, the cochaperone required for the loading of kinase client
% cell d eath

40
30
20
10
0 tion, which is crucial for chaperoning client proteins.14 Therefore,
control 3 µM Tubacin
Figure 6. Knockdown or inhibition of HDAC6 augments loss of clonogenic
survival due to treatment with 17-AAG. (A) K562-transfected with control/
siHDAC6 construct for 24 hours followed by treatment with indicated doses of
17-AAG for 24 hours and colonies were counted on day 10. Values plotted are
means of 3 experiments plus or minus SD. P values were determined by Student
t test. (B) K562-transfected with control/siHDAC6 construct for 48 hours, stained
with anti-HDAC6 and anti–Ki-67 antibodies, and imaged using confocal immuno-
fluorescent microscope using a 63⫻/1.2 W correction objective. (C) K562 cells
were treated with indicated doses of tubacin with or without 17-AAG for 48 hours,
and the loss of survival was assessed by trypan blue exclusion test. Error bars
represent SD.
proteins on to hsp90, remained unimpaired. It is recognized that
cdc37 differs from p23 in its ability to associate equally well with
both ATP-bound and 17-AAG–bound hsp90.33 Structural data
support the model that cdc37 can bind the open, ATP-free
conformation of the N-termini of the hsp90 dimer, and that ATP
binding converts the hsp90 dimer into a closed or tensed conforma-
1 µM 17-AAG 1 µM 17-AAG + although 17-AAG does not affect cdc37 binding to hsp90, it may
3 µM Tubacin prevent ATP-dependent chaperoning of client proteins by hsp90.14
Identification of specific acetylation site(s) that are involved in
the reversible hyperacetylation of hsp90 is critical in comprehen-
sively defining the role of this posttranslational modification in
chaperone function of hsp90. Recently, K294 has been identified as
one of the many acetylation sites that is at the junction of the
N-terminal and middle domain of hsp90.34,35 Acetylation-
mimetic lysine to glutamine mutant of K294 showed a decrease
in the client protein and cochaperone binding.34 Our recent
findings have indicated that additional functional acetylation

Table 1. Effect of tubacin and/or 17-AAG on viability of primary AML and CML-BC (blast crisis) blasts and normal CD34ⴙ progenitor cells
3 ␮M 1 ␮M 3 ␮M 3 ␮M tubacin plus 3 ␮M tubacin plus
Control tubacin 17-AAG 17-AAG 1 ␮M 17-AAG 3 ␮M 17-AAG

AML 1 7.6 12.7 26.6 31.3 37.3 47.4

AML 2* 9.4 13.4 36.8 49.5 52.0 59.6
AML 3* 2.7 8.1 24.3 39.4 31.5 47.8
CML 1 0.0 2.1 13.2 27.0 35.8 37.3
CML 2* 4.5 11.3 19.7 24.0 29.3 36.8
CML 3* 4.8 12.6 33.2 44.2 46.9 53.7
CD34⫹ normal† 7.9 ⫾ 1.8 13.8 ⫾ 0.4 16.5 ⫾ 0.9 20.3 ⫾ 0.9 21.2 ⫾ 2.2 16.4 ⫾ 2.5

Data are percentages of dead cells.

*CML and AML samples were grown on the human bone marrow–derived HS-5 stromal cells.
†CD34⫹ normal values are means plus and minus SD.
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
1892 RAO et al BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5

sites are located in the N-terminal and middle domain of hsp90,
which regulate the binding of ATP, cochaperone p23, and
17-AAG to hsp90.25,35 These observations highlight that, by
modulating its chaperone function, reversible hyperacetylation
of hsp90 provides another layer of control on the activity and
levels of the signaling hsp90 client proteins, as well as the
response of the cell to environmental stimuli.36
As inferred from our preferential displacement of biotinylated
GM-binding studies, hyperacetylation of hsp90 induced by siRNA
to HDAC6 increased the affinity of hsp90 for 17-AAG. This
augmented 17-AAG–mediated inhibition of ATP and cochaperone
binding. Enhanced affinity of 17-AAG for hsp90 also increased
17-AAG–mediated disruption of the chaperone function of hsp90
in K562 cells, resulting in greater polyubiquitylation and proteaso-
mal degradation of hsp90 client proteins, including Bcr-Abl, AKT,
and c-Raf. This was associated with the observation that the cells
cotreated with 17-AAG and the siRNA to HDAC6 showed greater
loss of clonogenic survival than due to either agent alone. Notably,
HDAC6 knockdown alone with resulting hyperacetylation of
hsp90 was sufficient to markedly reduce the colony-forming ability
of K562 cells. Associated with depletion of Ki67 expression, this
was not associated with induction of apoptosis or with alteration in
the percentage of cells in the S phase of the cell cycle. Although in
vitro treatment with tubacin was reported to not deplete BrdU-
positive S phase population or increase sub-G1 fraction of cells,27
this has not, as yet, been confirmed in the in vivo setting due to the
lack of availability of sufficient quantities of HDAC6-specific
inhibitor for the in vivo studies. It is tempting to speculate that
HDAC6 knockdown-mediated lysine hyperacetylation and alter-
ation of the function of proteins other than hsp90 may also
contribute to the increased loss of clonogenic survival of K562
cells.37 Inhibition of HDAC6 activity or depletion of HDAC6
levels also disrupts the protective handling of misfolded and
ubiquitylated toxic protein aggregates, which too can affect the
clonogenic survival of K562 cells.37 Cotreatment of K562 cells
with tubacin and 17-AAG resulted in enhanced loss of cell
survival, even though treatment with tubacin alone did not affect
the viability of K562 cells, as has also been noted in previous
studies.27 A recent report noted that mice lacking HDAC6 have
hyperacetylated tubulin but are viable and develop normally.38 It
would be important to demonstrate the in vivo response to 17-AAG
in these mice. In addition, it has been recently observed that normal
cytoplasmic HDAC6 function is required for efficient in vivo
oncogenesis in HDAC6 knockout mice (T. P. Yao, Duke University,
oral communication, May 19, 2008).
Present studies also show that HDAC6 binds and has chaperone
association with hsp90. This is supported by the observation that
treatment with 17-AAG shifted the binding of HDAC6 from hsp90
to hsp70, which was associated with the degradation of HDAC6 by
the 26S proteasome. Thus, by inhibiting chaperone function of
hsp90, 17-AAG also depletes HDAC6 levels. This explains why
17-AAG treatment was also associated with hyperacetylation of
hsp90 (Figure 4A). Because HDAC6 shuttles misfolded proteins
into the protective aggresome, 17-AAG–mediated depletion of
HDAC6 could undermine the protection against misfolded proteins
afforded by the aggresome.39 Boyault et al40 and Westerheide and
Morimoto41 have recently demonstrated that cellular stress and
increased levels of misfolded polyubiquitylated proteins caused by
treatment with a proteasome inhibitor trigger the dissociation of a
repressive HDAC6/hsp90/HSF1 (heat shock factor 1) complex,
leading to phosphorylation, trimerization, nuclear localization, and
transcriptional activity of HSF1. The latter induces the expression
of the major cellular chaperones. Dissociation of HDAC6 from
hsp90 also induces hsp90␣ acetylation. Collectively, these observa-
tions also explain why cotreatment with 17-AAG and the protea-
some inhibitor bortezomib results in proteotoxic ER stress, accen-
tuated by simultaneous depletion of HDAC6 along with inhibition
of the formation of the protective aggresomes.40,42 Thus, increased
ER stress noted after cotreatment with 17-AAG and bortezomib is
likely due to 17-AAG–mediated depletion of HDAC6.42-44 This
combination is currently being investigated against multiple my-
eloma, where ER stress is constitutively active and can be
accentuated to exert selective anti–multiple myeloma activity.43,44
In the present studies, we also show that targeted knockdown of
HDAC6 induces hyperacetylation of hsp70. This suggests that
HDAC6 also deacetylates hsp70. However, the mechanistic role
that hsp70 hyperacetylation plays in regulating 17-AAG–induced
disruption of the chaperone association of hyperacetylated hsp90
with its client proteins remains to be fully elucidated.
Acknowledgments
We wish to thank Drs Stuart Schreiber and Ralph Mazitschek, and
Initiative for Chemical Genetics, NIH (Bethesda, MD) for provid-
ing tubacin for the studies.
Authorship
Contribution: R.R. performed the experiments, evaluated the
results, and helped in writing the paper; W.F., Y.Y., P.L., R.J., P.F.,
and A.M. helped in performing the studies; P.A. provided reagents
and helped design the experiments; J.E.B. provided important
reagents for the studies; and K.B. helped design the experiments,
evaluated the data, and wrote the paper.
Conflict-of-interest disclosure: P.A. is an employee of Novartis
Institute for Biomedical Research, Inc. K.B. has received clinical
and laboratory research grant from Novartis Institute for Biomedi-
cal Research, Inc. All other authors declare no competing financial
interests.
Correspondence: Kapil Bhalla, Medical College of Georgia
Cancer Center, 1120 15th Street, CN2101A, Augusta, GA 30912;
e-mail: kbhalla@mcg.edu.

References
1. Pratt WB, Toft DO. Regulation of signaling protein
function and trafficking by the hsp90/hsp70-
based chaperone machinery. Exp Biol Med (May-
wood). 2003;228:111-133.
2. Neckers LJ. Heat shock protein 90: the cancer
chaperone. J Biosci. 2007;32:517-530.
3. Whitesell L, Lindquist SL. HSP90 and the chaper-
oning of cancer. Nat Rev Cancer. 2005;5:761-
772.
4. Ali MM, Roe S, Vaughan CK, et al. Crystal struc-
ture of an Hsp90-nucleotide-p23/Sba1 closed
chaperone complex. Nature. 2006;440:1013-
1017.
5. Shiau AK, Harris SF, Southworth DR, Agard D.
Structural analysis of E. coli hsp90 reveals dra-
matic nucleotide-dependent conformational rear-
rangements. Cell. 2006;127:329-340.
6. Meyer P, Prodromou C, Hu B, et al. Structural and
functional analysis of the middle segment of
hsp90: implications for ATP hydrolysis and client
protein and cochaperone interactions. Mol Cell.
2003;11:647-658.
7. Cintron N, Toft D. Defining the requirements for
Hsp40 and Hsp70 in the Hsp90 chaperone path-
way. J Biol Chem. 2006;281:26235-26244.
8. Roe SM, Ali MM, Meyer P, et al. The mechanism
of Hsp90 regulation by the protein kinase-specific
cochaperone p50(cdc37). Cell. 2004;116:87-98.
9. Grenert JP, Sullivan WP, Fadden P, et al. The
amino-terminal domain of heat shock protein 90
 From www.bloodjournal.org by on November 22, 2010. For personal use only.
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
(hsp90) that binds geldanamycin is an ATP/ADP
switch domain that regulates hsp90 conforma-
tion. J Biol Chem. 1997;272:23843-23850.
10. Nimmanapalli R, O’Bryan E, Kuhn D, Yamaguchi
H, Wang HG, Bhalla KN. Regulation of 17-AAG-
induced apoptosis: role of Bcl-2, Bcl-XL, and Bax
downstream of 17-AAG-mediated down-
regulation of Akt, Raf-1, and Src kinases. Blood.
2003;102:269-275.
11. Nimmanapalli R, O’Bryan E, Bhalla K. Geldana-
mycin and its analogue 17-allylamino-17-
demethoxygeldanamycin lowers Bcr-Abl levels
and induces apoptosis and differentiation of
Bcr-Abl-positive human leukemic blasts. Cancer
Res. 2001;61:1799-1804.
12. Bonvini P, Gastaldi T, Falini B, Rosolen A.
Nucleophosmin-anaplastic lymphoma kinase
(NPM-ALK), a novel Hsp90-client tyrosine kinase:
down-regulation of NPM-ALK expression and ty-
rosine phosphorylation in ALK(⫹) CD30(⫹) lym-
phoma cells by the Hsp90 antagonist 17-al-
lylamino,17-demethoxygeldanamycin. Cancer
Res. 2002;62:1559-1566.
13. George P, Bali P, Annavarapu S, et al. Combina-
tion of the histone deacetylase inhibitor LBH589
and the hsp90 inhibitor 17-AAG is highly active
against human CML-BC cells and AML cells with
activating mutation of FLT-3. Blood. 2005;105:
1768-1776.
14. Vaughan CK, Gohlke U, Sobott F, et al. Structure
of an Hsp90-Cdc37-Cdk4 complex. Mol Cell.
2006;23:697-707.
15. Peng C, Brain J, Hu Y, et al. Inhibition of heat
shock protein 90 prolongs survival of mice with
BCR-ABL-T315I-induced leukemia and sup-
presses leukemic stem cells. Blood. 2007;110:
678-685.
16. Bali P, Pranpat M, Bradner J, et al. Inhibition of
histone deacetylase 6 acetylates and disrupts the
chaperone function of heat shock protein 90: a
novel basis for antileukemia activity of histone
deacetylase inhibitors. J Biol Chem. 2005;280:
26729-26734.
17. Zhang X, Yuan Z, Zhang Y, et al. HDAC6 modu-
lates cell motility by altering the acetylation level
of cortactin. Mol Cell. 2007;27:197-213.
18. Kovacs JJ, Murphy PJ, Gaillard S, et al. HDAC6
regulates Hsp90 acetylation and chaperone-
dependent activation of glucocorticoid receptor.
Mol Cell. 2005;18:601-607.
19. Nimmanapalli R, Fuino L, Bali P, et al. Histone
deacetylase inhibitor LAQ824 both lowers ex-
pression and promotes proteasomal degradation
of Bcr-Abl and induces apoptosis of imatinib
mesylate-sensitive or -refractory chronic myelog-
enous leukemia-blast crisis cells. Cancer Res.
2003;63:5126-5135.
HYPERACETYLATION OF hsp90 AND 17-AAG ACTIVITY
20. Bali P, George P, Cohen P, et al. Superior activity
of the combination of histone deacetylase inhibi-
tor LAQ824 and the FLT-3 kinase inhibitor
PKC412 against human acute myelogenous leu-
kemia cells with mutant FLT-3. Clin Cancer Res.
2004;10:4991-4997.
21. Fiskus W, Pranpat M, Balasis M, et al. Histone
deacetylase inhibitors deplete enhancer of zeste
2 and associated polycomb repressive complex 2
proteins in human acute leukemia cells. Mol Can-
cer Ther. 2006;5:3096-3104.
22. Kamal A, Thao L, Sensintaffar J, et al. A high-
affinity conformation of Hsp90 confers tumour
selectivity on Hsp90 inhibitors. Nature. 2003;425:
407-410.
23. National Center for Biotechnology Information.
Entrez Nucleotide. http://www.ncbi.nlm.nih.gov/
GenBank. Accessed July 25, 2008.
24. Beer S, Zetterberg A, Ihrie RA, et al. Develop-
mental context determines latency of MYC-
induced tumorigenesis. PLoS Biol. 2004;2:1785-
1798.
25. Yang Y, Rao R, Shen J, et al. Role of acetylation
and extracellular location of heat shock protein
90␣ in tumor cell invasion. Cancer Res. 2008;68:
4833-4842.
26. Khan N, Jeffers M, Kumar S, et al. Determination
of the class and isoform selectivity of small-
molecule histone deacetylase inhibitors. Biochem
J. 2008;409:581-589.
27. Haggarty SJ, Koeller KM, Wong JC, Grozinger
CM, Schreiber SL. Domain-selective small-
molecule inhibitor of histone deacetylase 6
(HDAC6)-mediated tubulin deacetylation. Proc
Natl Acad Sci U S A. 2003;100:4389-4394.
28. Hideshima T, Bradner JE, Wong J, et al. Small-
molecule inhibition of proteasome and aggre-
some function induces synergistic antitumor ac-
tivity in multiple myeloma. Proc Natl Acad Sci
U S A. 2005;102:8567-8572.
29. Fiskus W, Ren Y, Mohaptra A, et al. Hydroxamic
acid analogue histone deacetylase inhibitors at-
tenuate estrogen receptor alpha levels and tran-
scriptional activity: a result of hyperacetylation
and inhibition of chaperone function of heat shock
protein 90. Clin Cancer Res. 2007;13:4882-4890.
30. Chen L, Meng S, Wang H, et al. Chemical abla-
tion of androgen receptor in prostate cancer cells
by the histone deacetylase inhibitor LAQ824. Mol
Cancer Ther. 2005;4:1311-1319.
31. Bali P, Pranpat M, Swaby R, et al. Activity of sub-
eroylanilide hydroxamic acid (SAHA) against hu-
man breast cancer cells with amplification of
Her-2. Clin Cancer Res. 2005;11:6382-6389.
32. McLaughlin SH, Sobott F, Yao Z, et al. The co-
chaperone p23 arrests the Hsp90 ATPase cycle
1893
to trap client proteins. J Mol Biol. 2006;356:746-
758.
33. Karnitz LM, Felts SJ. Cdc37 regulation of the ki-
nome: when to hold ’em and when to fold ’em. Sci
STKE. (http://stke. sciencemag.org/2007; pe22
(DOI 10.1126/stke.3852007pe22).
34. Scroggins BT, Robzyk K, Wang D, et al. An acety-
lation site in the middle domain of Hsp90 regu-
lates chaperone function. Mol Cell. 2007;25:151-
159.
35. Yang Y, Bhalla K. Identification and role of lysine
acetylation sites in regulating chaperone function
of heat shock protein 90␣. Proc Amer Assoc Can-
cer Res. 2007;48:170-171.
36. Aoyagi S, Archer TK. Modulating molecular chap-
erone Hsp90 functions through reversible acety-
lation. Trends Cell Biol. 2005;15:565-567.
37. Boyault C, Sadoul K, Pabion M, Khochbin S.
HDAC6, at the crossroads between cytoskeleton
and cell signaling by acetylation and ubiquitina-
tion. Oncogene. 2007;26:5468-5476.
38. Zhang Y, Kwon S, Yamaguchi T, et al. Mice lack-
ing histone deacetylase 6 have hyperacetylated
tubulin but are viable and develop normally. Mol
Cell Biol. 2008;28:1688-1701.
39. Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM,
Ito A, Yao TP. The deacetylase HDAC6 regulates
aggresome formation and cell viability in re-
sponse to misfolded protein stress. Cell. 2003;
115:727-738.
40. Boyault C, Zhang Y, Fritah S, et al. HDAC6 con-
trols major cell response pathways to cytotoxic
accumulation of protein aggregates. Genes Dev.
2007;21:2172-2181.
41. Westerheide SD, Morimoto RI. Heat shock re-
sponse modulators as therapeutic tools for dis-
eases of protein conformation. J Biol Chem.
2005;280:33097-33100.
42. Mimnaugh EG, Xu W, Vos M, et al. Simultaneous
inhibition of hsp 90 and the proteasome promotes
protein ubiquitination, causes endoplasmic
reticulum-derived cytosolic vacuolization, and
enhances antitumor activity. Mol Cancer Ther.
2004;3:551-566.
43. Davenport EL, Moore HE, Dunlop AS, et al. Heat
shock protein inhibition is associated with activa-
tion of the unfolded protein response pathway in
myeloma plasma cells. Blood. 2007;110:2641-
2649.
44. Hideshima T, Mitsiades C, Tonon G, Richardson
PG, Anderson KC. Understanding multiple my-
eloma pathogenesis in the bone marrow to iden-
tify new therapeutic targets. Nat Rev Cancer.
2007;7:585-598.