INHIBITION OF HISTONE DEACETYLASE 6 ACETYLATES AND DISRUPTS THE CHAPERONE FUNCTION OF HEAT SHOCK PROTEIN 90: A NOVEL BASIS OF

ANTILEUKEMIA ACTIVITY OF HISTONE DEACETYLASE INHIBITORS Purva Bali, Michael Pranpat, James Bradner, Maria Balasis, Warren Fiskus, Fei Guo, Kathy

Rocha, Sandhya Kumaraswamy, Sandhya Boyapalle, Peter Atadja, Edward Seto and Kapil Bhalla From the Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center, Tampa, FL, and Dana

Farber Cancer Institute, Boston, MA and Novartis Pharmaceuticals Inc. Cambridge, MA Running title: HDAC6 is a deacetylase for hsp90

Address for correspondence: Kapil Bhalla, M.D., Interdisciplinary Oncology Program, Moffitt Cancer Center and Research Institute, University of South Florida

12902 Magnolia Drive, MRC 3 East, Room 3056, Tampa, FL 33612, U.S.A., Tel. 813-903-6861; Fax. 813-903-6817; E-mail: bhallakn@moffitt.usf.edu

The hydroxamic acid (HAA) analogue

pan-histone deacetylase (HDAC) inhibitors (HDIs) LAQ824 and LBH589 have been shown to induce acetylation and inhibit the ATP binding and chaperone function of heat shock protein (hsp) 90. This promotes the polyubiquitylation and degradation of the pro-growth and pro-survival client proteins Bcr-Abl, mutant FLT-3, c-Raf and AKT in human leukemia cells. HDAC6 is a member of the class IIB of HDACs. It is predominantly cytosolic, microtubule associated, α-tubulin deacetylase, which is also known to promote aggresome inclusion of the misfolded polyubiquitylated proteins. Here we demonstrate that, in the Bcr-Abl oncogene expressing human leukemia K562 cells, HDAC6 can be co-immunoprecipitated with hsp90, and the knockdown of HDAC6 by its siRNA induced the acetylation of hsp90 and α-tubulin. Depletion of HDAC6 levels also inhibited the binding of hsp90 to ATP, reduced the chaperone association of hsp90 with its client proteins, e.g., Bcr-Abl, as well as induced polyubiquitylation and partial depletion of Bcr-Abl. Conversely, the ectopic overexpression of HDAC6 inhibited LAQ824-induced acetylation of hsp90 and α-tubulin, as well as reduced LAQ824 mediated depletion of Bcr-Abl, AKT and c-Raf. Collectively, these findings indicate that HDAC6 is also an hsp90 deacetylase. Targeted inhibition of HDAC6 leads to acetylation of hsp90 and disruption of its chaperone function. This results in polyubiquitylation and depletion of pro-growth and pro-survival hsp90 client proteins

including Bcr-Abl. By also inhibiting the inclusion of the misfolded and polyubiquitylated proteins into the aggresome, the depletion of HDAC6 sensitizes human leukemia cells to HAA-HDIs and proteasome inhibitors.

Several reports have highlighted that the heat

shock protein (hsp) 90 serves as the chaperone protein, required for the proper folding and maintenance of a number of signaling protein kinases, e.g., the wild type or mutant Bcr-Abl, mutant FLT-3, AKT and c-Raf, in their native, mature, stable and functionally active conformation (1-6). This is achieved by hsp90 through ATP binding and hydrolysis (7,8). ATP/ADP binding to the hydrophobic N-terminus pocket alters hsp90 conformation, resulting in the interaction of hsp90 with a co-chaperone complex that protects or stabilizes the client proteins, or with an alternative subset of co-chaperones that directs the misfolded client proteins to a covalent linkage with polyubiquitin and subsequent degradation by the 26S proteasome (7-9). Benzoquinone ansamycin antibiotic geldanamycin (GA) and its less toxic analogue 17-AAG bind to the ATP/ADP binding pocket, replacing the nucleotide and inhibiting the chaperone function of hsp90 (3,6,10). By blocking ATP binding to hsp90, treatment with 17-AAG stabilizes hsp90 conformation into the alternative one that recruits hsp70-based co-chaperone complex, which binds the misfolded client proteins and directs them to polyubiquitylation and proteasomal degradation (11).

HATs (histone acetyl transferases) and HDACs

(histone deacetylases) are enzymes that catalyze the acetylation and deacetylation, respectively, of the N-

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termini of the core nucleosomal histone tails at evolutionarily conserved lysine residues (12-14). Following recruitment by multiprotein transcriptional complexes to the promoters of genes, the balance between the activities of HATs and HDACs regulates the acetylation status of chromatin and transcription through chromatin modification without directly binding the DNA (15). Importantly, these enzymes also affect the acetylation status of specific lysine residues of a number of transcription factors, which may affect their DNA binding and transcriptional activity (16,17). Three classes of HDACs have been identified, which include class I, II (A & B) and III (16,17). Treatment with the novel hydroxamic acid analogue (HAA) pan-HDAC inhibitors (HDIs) LAQ824 and LBH589 were shown not only to induce acetylation of histones, induction of p21, cell cycle growth arrest and apoptosis, but also demonstrated to induce acetylation of hsp90 (18-20). This was associated with polyubiquitylation, proteasomal degradation and depletion of Bcr-Abl, AKT and c-Raf in CML, as well as mutant FLT-3 in AML cells (18-20). These observations raised the issue as to which of the HDACs is responsible for deacetylating hsp90, such that its inhibition by HAA-HDIs induces acetylation and undermines the chaperone function of hsp90. HDAC6 is predominantly a cytoplasmic, microtubule associated member of the class IIB family of HDACs (21). HDAC6 is unique in possessing two catalytic domains and a C-terminal zinc finger domain that binds both polyubiquitylated misfolded proteins and the dynein motor (22,23). The C-terminus catalytic domain of HDAC6 possesses α-tubulin deacetylase activity, which is inhibited by the small molecule inhibitor tubacin (24-27). HDAC6 has also been demonstrated to direct the polyubiquitylated misfolded proteins into aggresomes, thus regulating the cellular management of the misfolded protein stress response (23). Collectively, these features of HDAC6 suggested that HDAC6 might be involved in hsp90 deacetylation. In the present studies we determined whether HDAC6 regulates the acetylation status of hsp90, and whether depletion or inhibition of HDAC6

affects the chaperone function of hsp90 in human leukemia cells.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies. 17-AAG was

obtained from Developmental Therapeutics Branch of CTEP/NCI/NIH (Bethesda, MD). LBH589 and LAQ824 were provided by Novartis Pharmaceuticals Inc. (East Hanover, NJ). ATP-Binders Resin was purchased from EMD Biosciences (San Diego, CA). Anti-hsp90 and -hsp70 antibodies were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Monoclonal anti-acetyl lysine and polyclonal anti-HDAC3 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal anti-acetyl α-tubulin and anti-FLAG (M2) antibody were purchased from Sigma Aldrich Corporation (St. Louis, MO). Monoclonal anti-Abl and HDAC1, and polyclonal anti-HDAC2 and HDAC6 were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA). Polyclonal HDAC10 antibody was purchased from Biovision Inc. (Mountainview, CA).

Cell Culture. Human CML K562 (expressing

Bcr-Abl), acute myeloid leukemia HL-60 and acute leukemia MV4-11 (containing a 30 base pair long ITD in the exon 14 of FLT-3) cells were obtained from American Tissue Culture Collection (Manassas, VA) and maintained in culture in the RPMI medium containing 10% fetal bovine serum. The cells were passaged twice a week, as previously described (19,28). Logarithmically growing cells were exposed to the designated concentrations and exposure interval of the drugs. Following these treatments, cells were pelleted and washed free of the drug(s) prior to the performance of the studies described below.

Western Blot Analyses. Western analyses of

Bcr-Abl, c-Raf-1, AKT, p21, HDAC1, 2, 3, 6 and 10 proteins and β-actin were performed using specific anti-sera or monoclonal antibodies (see above), as described previously (19,28,29). Horizontal scanning densitometry was performed on Western blots by using acquisition into Adobe PhotoShop (Adobe Systems Inc., San Jose, CA) and analysis by the NIH Image Program (U.S. National Institutes of

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Health, Bethesda, MD). The expression of β-actin was used as a loading control.

Acetylation of Hsp90 and its binding to

ATP-sepharose: Untreated or LAQ824 treated cells were lysed in TNESV buffer (50 mM Tris, 2 mM EDTA, 100 nM NaCl, 1 mM sodium orthovanadate, 25 mM NaF and 1% Triton X-100 at pH 7.5), and total cellular proteins were quantified using the BCA protein assay. Hsp90 was immunoprecipitated from 200 µg of total protein using mouse hsp90 antibody, and immunoprecipitates were immunoblotted with anti-acetyl lysine antibody, as previously described (18,19,30). Alternatively, cell lysates were prepared after drug treatment and hsp90 was affinity precipitated from 200 µg of total cellular protein using ATP–sepharose beads. The concentration of hsp90 in the precipitates was assessed by Western blot analysis using a monoclonal anti-hsp90 antibody (18,19,30).

Assessment of Percentage Non-viable

cells: Cells were stained with trypan blue (Sigma, St. Louis, MO). The numbers of non-viable cells were determined by counting the cells that showed trypan blue uptake in a hemocytometer, and reported as percentage of untreated control cells (5,20).

Immunoprecipitation of hsp90 or Bcr-

Abl and Immunoblot Analyses: Following the designated treatments, cells were lysed in the lysis buffer (20 mM Tris (pH 8), 150 nM sodium chloride, 1% Triton X-100, 0.1 M NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 2.5 µg /ml leupeptin, 5 µg /ml aprotinin) for 30 minutes on ice, and the nuclear and cellular debris cleared by centrifugation. Cell lysates (200 µg) were incubated with the hsp90 or Abl-specific monoclonal antibody for one hour at 4°C. To this, washed Protein G agarose beads were added and incubated overnight at 4°C. The immunoprecipitates were washed three times in the lysis buffer and proteins were eluted with the SDS sample loading buffer prior to the immunoblot analyses with specific antibodies against hsp90, anti-abl, HDAC6, anti-acetyl lysine or anti-ubiquitin antibody (18,31).

Preparation of detergent-soluble and insoluble fractions: After the designated drug treatments, cells were lysed with TNSEV buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate, 1% NP-40 containing 20 µg/ml aprotonin, 20 µg/ml leupeptin, 1 mM PMSF, 25 mM NaF, and 5 mM N-ethylmaleimide) (16). The insoluble fraction (pellet) were solubilized with SDS buffer (80 mM Tris, pH 6.8, 2% SDS, 100 mM DTT, and 10% glycerol). Fifty µg of proteins from the NP-40 soluble and insoluble fractions were separated on 7.5% SDS-polyacrylamide gel and analyzed by Western blotting (18,19).

Ectopic overexpression of HDAC6: N-

terminus FLAG-tagged cDNA of HDAC6 (kindly provided by Dr. Eric Verdin, Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA) was cloned into the pcDNA3.1 plasmid vector (Invitrogen Corp.Carlsbad, CA). This and the control vector were transiently transfected into K562 cells by a previously described method (18,32).

Creation and transfection of HDAC6 siRNA

vector: K562 cells were transiently transfected utilizing Amaxa Nucleofector Electroporator with the plasmid vector pBS/U6 with or without the HDAC6 siRNA (32). This encoded a 21-nucleotide sequence 5’ GG ATG GAT CTG AAC CTT GAG A 3’, corresponding to the targeted codons 200-219 in the HDAC6 mRNA (Accession # BC013737), or a non-specific siRNA encoding sequence. Forty-eight hours following transfection, cell lysates were screened for HDAC6 expression by Western blot analysis. The most efficient knockdown cells with markedly reduced expression of HDAC6 were utilized for further studies.

Statistical Analyses. Data were expressed as

mean ± SEM. Comparisons used student’s t test or ANOVA, as appropriate. P values of < 0.05 were assigned significance.

RESULTS

We first determined the effects of LAQ824 and

LBH589 treatment on hsp90 acetylation in the human leukemia K562, MV4-11 and HL-60 cells. Following exposure to 100 or 250 nM LAQ824 for 16 hours, immunoprecipitation of hsp90 followed by

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immunobotting with an antibody that recognizes anti-acetylated lysine residues was performed. Acetylation of hsp90 in a dose dependent manner was observed, without significant effect on the levels of hsp90 (Figure 1A). Exposure to LBH589 exerted similar effect on hsp90 acetylation (data not shown). In contrast, treatment with sodium butyrate or trapoxin for 16 hours, which are known not to inhibit the activities of class II HDACs, did not induce hsp90 acetylation (Figure 1B). Exposure to tubacin, which inhibits only the α-tubulin deacetylase (TDAC) domain of HDAC6, caused only minimal increase in hsp90 acetylation. Following treatment of K562 cells with LAQ824 or LBH589 for 16 hours, hsp90 was affinity precipitated from the cell lysates with ATP-sepharose, and the levels of hsp90 were estimated by immunoblot analysis. LAQ824 and LBH589 mediated acetylation of hsp90 was associated with decreased binding of ATP sepharose to hsp 90 (Figure 1C). While 17-AAG, the geldanamycin analogue inhibitor of the ATP binding pocket of hsp90, was more effective in inhibiting the binding of ATP sepharose to hsp90, treatment with sodium butyrate, trapoxin or tubacin (not shown) was ineffective (Figure 1C).

Next, we evaluated the impact of these

modifications on the function of hsp90 as a chaperone protein. Following exposure of K562 cells to LAQ824 for 16 hours, Bcr-Abl was immunoprecipitated from the cell lysates, and the binding of Bcr-Abl to hsp90 versus hsp70 was determined. Figure 2A demonstrates that, similar to what has been observed with 17-AAG, treatment with LAQ824 shifted the chaperone association of Bcr-Abl from hsp90 to hsp70. A similar shift in the chaperone association was also seen following treatment with LBH589 (data not shown). Previous reports have shown that the shift in the chaperone association of the client proteins to the hsp70 containing unstable multichaperone complex induces polyubiquitylation and accumulation of the client proteins in the detergent-insoluble cellular fraction. This marks the client proteins for degradation by the 26S proteasome (3,6). Consistent with this, treatment with LAQ824 increased Bcr-Abl accumulation in the

detergent-insoluble fraction, which was further enhanced by co-treatment with the proteasome inhibitor PS341 (bortezomib) (Figure 2B). Additionally, treatment of K562 cells with LAQ824 increased the polyubiquitylation of proteins in the Bcr-Abl-containing immunoprecipitates with anti-Abl antibody (Figure 2B). Increased polyubiquitylation of proteins was also seen, following treatment with PS341, or following co-treatment with LAQ824 and PS341 (Figure 2B). Consistent with this, treatment with LAQ824 depleted the levels of Bcr-Abl in K562 cells (Figure 2C). As also previously reported, LAQ824 treatment depleted c-Raf and AKT levels (18,19), while concomitantly inducing the levels of the acetylated α-tubulin and p21 in a dose dependent manner (Figure 2C).

Simultaneous induction of the acetylation of α-

tubulin and hsp90 by LAQ824 raised the possibility that inhibition of HDAC6 may be responsible for LAQ824-mediated acetylation and inhibition of the chaperone function of hsp90. To address this issue, we first determined whether HDAC6 is co-immunoprecipitated with hsp90. As shown in Figure 3A, in K562 cells HDAC6 could be co-immunoprecipitated with hsp90. Treatment with LAQ824 or LBH589 for 16 hours, but not with sodium butyrate, reduced the amount of HDAC6 that could be co-immunoprecipitated with hsp90, as estimated by densitometry (Figure 3A). LAQ824 but not trapoxin treatment was associated with slight increase in the levels of HDAC6, while neither drug significantly affected the levels of HDAC10 (most closely related HDAC to HDAC6) (data not shown). We next determined the effect of the ectopic overexpression of HDAC6 on LAQ824-induced acetylation of α-tubulin and hsp90 and on induction of p21 in K562 cells. A FLAG-tagged cDNA of HDAC6 was transfected in K562 cells, and the transient expression of FLAG and the overexpression of HDAC6 was confirmed in K562/HDAC6 cells (Figure 3B). As compared to the vector-alone transfected control K562 cells, a two-fold increase in HDAC6 expression was detected in K562/HDAC6 cells. The discrepancy between FLAG expression and only a two-fold overexpression of HDAC6 may be because the anti-HDAC6 antibody poorly recognized its epitope in the FLAG-tagged version of HDAC6. No significant change in the HDAC10 levels was observed in

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K562/HDAC6 cells (Figure 3B). As compared to the control K562, LAQ824-mediated increase in the levels of acetylated α-tubulin but not of p21 was blunted in K562/HDAC6 cells (Figure 3B). LAQ824 treatment did not significantly affect HDAC6 or HDAC10 expressions in K562/HDAC6 and K562/control cells. Importantly, ectopic overexpression of HDAC6 also inhibited LAQ824-induced hsp90 acetylation in K562/HDAC6 cells (Figure 3C). Concomitantly, as compared to the effect of LAQ824 in K562/control cells, a lesser LAQ824-mediated attenuation of Bcr-Abl and c-Raf levels was observed in K562/HDAC6 cells (Figure 3D).

To demonstrate further that HDAC6 acts as

a deacetylase not only for α-tubulin but also for hsp90, we introduced a siRNA to HDAC6 into K562 cells to inhibit the expression of HDAC6. As shown in figure 4A, compared to the vector or non-specific siRNA transfected K562/Control, in the HDAC6-siRNA transfected K562/HDAC6-siRNA cells, HDAC6 levels were knocked down by approximately 50%, while HDAC3 or HDAC10 were not affected. As previously reported, the siRNA to HDAC6 induced the acetylation of α-tubulin in K562 cells (23,24). This was similar to the effect of treatment with LAQ824. Additionally, as is seen following hsp90 inhibition by 17-AAG, treatment with LAQ824 or HDAC6-siRNA also increased hsp70 levels (Figure 4A). Notably, treatment with HDAC6-siRNA also induced the acetylation of hsp90, without affecting the levels of hsp90 (Figure 4B). Thus, HDAC6 is a deacetylase not only for α-tubulin but also for hsp90. Moreover, hsp90 acetylation induced by HDAC6 inhibition attenuated the binding of ATP to hsp90, as is also seen following treatment with LAQ824 (Figure 4B) or 17-AAG (4,5). This suggested that the hsp90 acetylation in K562/HDAC6-siRNA cells disrupts the chaperone function of hsp90, a conclusion also supported by the induction of hsp70 presumably due to decreased association of heat shock factor 1 (HSF1) with hsp90 in K562/HDAC6-siRNA cells (6,33,34). Similar to treatment with 17-AAG or LAQ824, acetylation and inhibition of hsp90 function due to HDAC6-siRNA, attenuated the chaperone association of Bcr-Abl

with hsp90 (Figure 4C). As shown in Figure 4D, this was associated with increased polyubiquitylation of Bcr-Abl and depletion of the intracellular levels of Bcr-Abl, c-Raf and AKT, most likely through the proteasome (4,18). The variable extent of depletion of the client proteins is presumably due to differences in the levels of mRNA transcripts and their translation, rates of polyubiquitylation by the specific E3 ubiquitin ligase for the client protein, and/or the rates of degradation of the client proteins by the proteasome. In a previous report, it was demonstrated that cells deficient in HDAC6 are hypersensitive to the accumulation of misfolded proteins (23). Therefore, we compared the cytotoxic effects of polyubiquitylated and misfolded proteins induced by treatment of the control K562 and K562/HDAC6-siRNA cells with LAQ824 and/or bortezomib. Figure 4E demonstrates that treatment with LAQ824 and/or bortezomib induced more lethality in K562/HDAC6-siRNA versus the K562/control cells. This indicates that the abrogation of the protective effect afforded by HDAC6-mediated aggresome formation sensitized K562/HDAC6-siRNA cells more than K562/control cells to the lethal effects of LAQ824 or bortezomib. Consistent with this, combined inhibition of hsp90 and the proteasome due to co-treatment with LAQ824 and bortezomib, which markedly increases the intracellular content of misfolded and polyubiquitylated proteins in the cells (34,35), also exerted more lethal effects in the HDAC6-siRNA transfected versus the control K562 cells (Figure 4E).

DISCUSSION

It has been previously demonstrated that

treatment with the HAA-HDIs LAQ824 and LBH589 inhibits the chaperone function of heat shock protein (hsp) 90, promoting poly- ubiquitylation and degradation of the pro-growth and pro-survival Bcr-Abl, c-Raf and AKT proteins in human leukemia cells (18,20,36). Here we demonstrate for the first time that HDAC6 is the deacetylase for hsp90, and depletion of HDAC6 levels and activity is likely to be responsible for LAQ824 and LBH589 mediated acetylation and inhibition of hsp90 as a chaperone protein. The role of HDAC6 in mediating hsp90 deacetylation was strongly suggested when treatment with trapoxin and sodium butyrate, which do not inhibit HDAC6, did

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not induce the acetylation or disruption of ATP binding of hsp90. It was also suggested when tubacin, which is a specific inhibitor of only the TDAC domain of HDAC6 and is unable to inhibit the entire catalytic activity of HDAC6, only slightly induced the acetylation of hsp90. LAQ824 and LBH589 are pan-HDAC inhibitors that not only inhibit the class I and IIA but also class IIB HDACs, as was suggested by the increased acetylation of α-tubulin, a substrate for the predominantly cytosolic HDAC6 belonging to the class IIB family. This too supported the involvement of HDAC6 as a deacetylase for hsp90. Additionally, the ectopic overexpression of HDAC6 diminished LAQ824 mediated acetylation of not only α-tubulin but also of hsp90. It is noteworthy that HDAC6 overexpression did not inhibit LAQ824 mediated p21 induction, since this would be due to inhibition of HDAC1 by LAQ824. The findings presented here also show that HDAC6 is co-immunoprecipitated with hsp90, and this association is inhibited following treatment with LAQ824 or LBH589 but not sodium butyrate. This again strongly supported the role of the catalytic active HDAC6 in binding and deacetylating hsp90. The catalytic activity was also shown to be required for binding of HDAC6 to the dynein motor and misfolded proteins. It was also shown to be necessary for the aggresome formation (23). Finally, the demonstration that the specific knockdown of HDAC6 alone induces acetylation of hsp90 confirmed the role of HDAC6 as an hsp90 deacetylase.

Data presented here also demonstrate that

whether through specific knockdown of HDAC6 or inhibition of its catalytic activity by HDIs, increased acetylation of hsp90 has functional consequences. First, the induction of acetylation of hsp90 reduced the binding of hsp90 to ATP, although this appeared to be a less potent effect than seen following treatment with 17-AAG. Second, it reduced the chaperone association of hsp90 with its client proteins Bcr-Abl, which promoted its polyubiquitylation and accumulation in the detergent insoluble fraction. This was further enhanced by co-treatment with bortezomib. Finally, consistent with this, acetylation of hsp90 induced by HDAC6

inhibition or depletion attenuated the levels of client proteins, e.g., Bcr-Abl. Importantly, this indicates that a targeted inhibition of HDAC6 may lead to the depletion of specific pro-growth and pro-survival, leukemia associated, hsp90 client proteins through the disruption of the chaperone function of hsp90. Why each client protein is depleted to a different extent may be dependent on the activity of the different E3 ubiquitin ligase or the de-ubiquitylase required for the client proteins, as well as the half-life and intracellular abundance of the client protein. It should be noted that the present studies did not determine whether and how acetylation of hsp90 affects the association of hsp90 with its co-chaperones, which is known to be regulated by whether hsp90 is in the ATP or ADP bound state (2,37,38). It would also be clearly important to determine the lysine residues on hsp90 that are involved in HDAC6 mediated deacetylation, and the HAT involved in mediating acetylation of hsp90. Our current studies are focused on resolving these issues.

Present findings, demonstrating that the ectopic

overexpression and activity of HDAC6 diminished while treatment with HDAC6-siRNA induced the acetylation of α-tubulin are consistent with previous reports that α-tubulin is a substrate for the activity of TDAC domain of HDAC6 (23-25). HDAC6 has also been shown to interact with β-tubulin (25). While HDAC6-mediated tubulin deacetylation has been shown to delay tubulin depolymerization, acetylation of α-tubulin promoted tubulin polymerization, interaction with microtubule associated proteins and cell motility (26,27). Thus inhibition of HDAC6 or specific inhibition of its TDAC domain may mediate the antimetastatic and antiangiogenic effects of HAA-HDIs (27). It was earlier reported that the combination of HAA-HDI and 17-AAG induced increased intracellular levels of misfolded and polyubiquitylated proteins and exerted synergistic cytotoxic effects against Bcr-Abl or FLT-3 ITD expressing acute leukemia cells (36). Additionally, the combination of HAA-HDI with bortezomib has also been shown to exert synergistic in vitro cytotoxic effects (39). In the present studies, we have demonstrated that, similar to HDAC6 inhibition by HAA-HDIs, depletion of HDAC6 levels by HDAC6-siRNA also increases the lethal effects of bortezomib. This confirms an earlier report where inhibition of aggresome formation by

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HDAC6-siRNA resulted in decreased clearance of misfolded proteins, impaired aggresome formation and hypersensitivity to the cellular stress induced by misfolded proteins (23). Collectively, these observations support the concept that aggresome formation is an integral part of a protective response to misfolded proteins and HDAC6 plays a critical role in the management of the misfolded protein-induced stress response (23,40). In this context, it is noteworthy that, in patients with breast cancer,

the cytosolic expression of HDAC6 was correlated with hormone responsiveness and better survival (41). Whether small molecule drugs that specifically inhibit HDAC6 would sensitize cancer cells to the stress of misfolded proteins created by hsp90 and/or proteasome inhibitors remains to be established. It would be important to investigate the efficacy of such a combination against tumor cell types where protein turnover is an integral part of the molecular phenotype and cell survival, as in B cell malignancies and multiple myeloma.

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FIGURE LEGENDS

Fig. 1. LAQ824 treatment induces acetylation of hsp90, inhibits its binding to ATP. A, MV4-11, HL-60 and K562 cells were exposed to the indicated concentrations of LAQ824 for 16 hours. Following this, hsp90 was immunoprecipitated from the cell lysates and immunoblotted with either anti-hsp90 or anti-acetylated lysine antibody. B, K562 cells were treated with the HAA-HDI LAQ824 or LBH589 (that are able to inhibit HDAC6), or sodium butyrate or trapoxin (that do not inhibit HDAC6) for 16 hours. Following this, hsp90 immunoprecipitates were immunoblotted with anti-acetyl lysine or anti-hsp90 antibody. C, K562 cells were treated with the indicated concentrations of the drugs for 16 hours. Following this, hsp90 from the cell lysates was affinity precipitated with ATP-sepharose and subjected to immunoblotting with anti-hsp90 antibody.

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Fig. 2. LAQ824 shifts the chaperone association of Bcr-Abl from hsp90 to hsp70, resulting in polyubiquitylation and proteasomal degradation of Bcr-Abl. A, K562 cells were treated with the indicated concentration of LAQ824 for 16 hours. Following this, immunoprecipitates of Bcr-Abl from the cell lysates were immunoblotted with anti-Hsp90, anti-Hsp70 or anti-Abl antibodies. B, K562 cells were treated with the indicated concentrations of LAQ824 and/or PS341 (bortezomib) for 16 hours. Following this, immunoprecipitates of Bcr-Abl from the cell lysates were immunoblotted with anti-ubiquitin or anti-Abl antibodies. Additionally, following treatment of K562 cells with LAQ824 and/or PS341, the detergent (NP40)–insoluble fractions were obtained and immunoblotted with anti-Abl antibody. The levels of β-actin served as the loading control. C, K562 cells were treated with the indicated concentrations of LAQ824 for 16 hours. Following this, the cell lysates were immunoblotted with anti-acetyl α-tubulin, p21, Abl, c-Raf or anti-AKT antibody. The levels of β-actin served as the loading control. Fig. 3. HDAC6 binds to hsp90 and ectopic overexpression of HDAC6 inhibits LAQ824-mediated acetylation of α-tubulin and hsp90. A, K562 cells were treated with the indicated concentrations of the HAA-HDI LAQ824 or LBH589, or sodium butyrate for 16 hours. Following this, total cell lysates were prepared and immunoprecipitated to hsp90 or the control rat IgG. Immunoblot analysis was performed with either anti-hsp90 or anti-HDAC6 antibody. B, K562 were transiently transfected with pCDNA3.1 vector containing FLAG-tagged HDAC6 (K562/HDAC6 cells) or the vector alone (K562/Control) over 48 hours. Following this, the cells were treated with 250 nM of LAQ824 for 16 hours. After this treatment, the cell lysates were immunoblotted with anti-FLAG, HDAC6, HDAC10, acetyl α-tubulin or anti-p21 antibody. The levels of β-actin served as the loading control. C, K562/Control and K562/HDAC6 cells were treated with 250 nM LAQ824 for 16 hours. Following this treatment, immunoprecipitates of hsp90 were immunoblotted with anti-acetyl lysine or anti-hsp90 antibody. D. K562/Control and K562/HDAC6 cells were treated with 250 nM LAQ824 for 16 hours. Following this treatment, the cell lysates were immunblotted with anti-Abl or anti-c-Raf antibody. The levels of β-actin served as the loading control. Fig. 4: Depletion of HDAC6 induces acetylation and inhibits chaperone function of hsp90. A, K562 cells were transfected with the pBS/U6 vector containing the nucleotides that encode for the siRNA to HDAC6 over 48 hours. Following this, the cell lysates from K562 cells expressing HDAC6 siRNA or from untreated or LAQ824-treated K562 vector control cells were immunoblotted with anti-HDAC6, HDAC10, HDAC3, acetyl α-tubulin or anti-hsp70 antibody. The levels of β-actin served as the loading control. B, Alternatively, the cell lysates, as above, were utilized to immunoprecipitate hsp90, which was either immunoblotted with anti-acetyl lysine or anti-hsp90 antibody, or the cell lysates were affinity precipitated with ATP-sepharose and immunoblotted with anti-hsp90 antibody. C, The cell lysates from K562 cells expressing HDAC6 siRNA or from untreated or LAQ824-treated K562 vector control cells were immunoprecipitated with anti-Abl antibody, and the immunoprecipitates were either immunoblotted anti-hsp90 or anti-Abl antibody. D, The cell lysates from K562 cells expressing HDAC6 siRNA or from untreated or LAQ824-treated K562 control cells were immunoprecipitated with either with the control mouse IgG or anti-Abl antibody, and the immunoprecipitates were either immunoblotted with anti-ubiquitin or anti-Abl antibody. Alternatively, the cell lysates were immunoblotted with anti-Abl, AKT or anti-c-Raf antibody. The levels of β-actin served as the loading control. E, K562 cells expressing HDAC6 siRNA or K562 control cells were treated with 5 nM bortezomib, 250 nM LAQ824 or a combination of bortezomib and LAQ824 for 48 hours. Following this, the viability of cells was determined by trypan blue exclusion method. The mean percentage values (of three experiments) for non-viable cells following each treatment are represented as bar graphs.

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BhallaRocha, Sandhya Kumaraswamy, Sandhya Boyapalle, Peter Atadja, Edward Seto and Kapil

Purva Bali, Michael Pranpat, James Bradner, Maria Balasis, Warren Fiskus, Fei Guo, Kathyinhibitors

heat shock protein 90: A novel basis of antileukemia activity of histone deacetylase Inhibiition of histone deacetylase 6 acetylates and disrupts the chaperone function of

published online June 2, 2005J. Biol. Chem. 

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