Regulation of STAT3 by Histone Deacetylase-3 in Diffuse Large B-Cell Lymphoma: Implications for Therapy

ORIGINAL ARTICLE Regulation of STAT3 by histone deacetylase-3 in diffuse large B-cell lymphoma: implications for therapy

M Gupta1, JJ Han, M Stenson, L Wellik and TE Witzig1

Diffuse large B-cell lymphoma (DLBCL) with an activated B-cell (ABC) gene-expression proﬁle has been shown to have a poorer prognosis compared with tumors with a germinal center B-cell type. ABC cell lines have constitutive activation of STAT3; however, the mechanisms regulating STAT3 signaling in lymphoma are unknown. In studies of class-I histone deacetylase (HDAC) expression, we found overexpression of HDAC3 in phospho STAT3-positive DLBCL and the HDAC3 was found to be complexed with STAT3. Inhibition of HDAC activity by panobinostat (LBH589) increased p300-mediated STAT3Lys685 acetylation with increased nuclear export of STAT3 to the cytoplasm. HDAC inhibition abolished STAT3Tyr705 phosphorylation with minimal effect on STAT3Ser727 and JAK2 tyrosine activity. pSTAT3Tyr705-positive DLBCLs were more sensitive to HDAC inhibition with LBH589 compared with pSTAT3Tyr705-negative DLBCLs. This cytotoxicity was associated with downregulation of the direct STAT3 target Mcl-1. HDAC3 knockdown upregulated STAT3Lys685 acetylation but prevented STAT3Tyr705 phosphorylation and inhibited survival of pSTAT3-positive DLBCL cells. These studies provide the rationale for targeting STAT3-positive DLBCL tumors with HDAC inhibitors.

Leukemia (2012) 26, 1356--1364; doi:10.1038/leu.2011.340; published online 25 November 2011 Keywords: lymphoma; HDAC3; LBH589; STAT3; Mcl-1

INTRODUCTION HDAC9 and HDAC10); and the class-III SIR2-like proteins.11 Class-I Gene expression profiling studies have distinguished three HDACs are widely expressed, whereas the expression of many molecular subtypes of diffuse large B-cell lymphoma (DLBCL) class-II HDACs are tissue-specific. HDAC1, 2 and 8 are predomi- known as ‘germinal center B-cell-like’ (GCB), ‘activated B-cell-like’ nantly nuclear proteins, whereas HDACs 3, 4, 5, 7 and 9 shuttle 12 (ABC)1 and primary mediastinal B-cell type.2,3 GCB DLBCLs arise between the nucleus and cytoplasm. Different studies have from normal germinal center B cells, whereas ABC DLBCLs recently demonstrated that histones are not the only proteins 13 originate from post-germinal center B cells that are arrested under the control of HDACs. For example, HDACs can act on during plasmacytic differentiation.2 In general, the GCB group non-histone substrates, such as tubulin and the transcription 14,15 responds better to conventional chemotherapy, whereas patients factors p53 and NF-kB. Increasing evidence suggests that the with ABC type tend to be more refractory.4 Compared with the regulation of gene expression by STATs requires HDAC activity. current understanding of GCB-type DLBCL, the biology and The HDAC inhibitor tricostatin A inhibits STAT3-dependent pathogenic mechanisms of ABC-type DLBCL are less understood. transcriptional activity induced by platelet-derived growth factor The signal transducers and activators of transcription (STATs) in NIH 3T3 cells and blocks STAT1 activation in interferon gamma- 16 belong to a family of cytoplasmic transcription factors. Phospho- stimulated carcinoma cells. In this present study, we investigated rylated STATs form homo- or heterodimers that then translocate the molecular mechanism of STAT3 regulation in DLBCL and to the nucleus, where they bind DNA and regulate gene demonstrate that targeting pSTAT3-positive DLBCL with HDAC transcription.5,6 Phosphorylation and acetylation are crucial inhibitors is a potential therapeutic option for these patients. posttranslational modifications that regulate the activities of various proteins.7 With respect to lymphoma, NF-kB along with STAT3 is activated and highly expressed in ABC DLBCL cell lines.8,9 MATERIALS AND METHODS The mechanism of NF-kB regulation has been extensively studied Reagents in DLBCL, whereas the mechanisms that regulate STAT3 in these LBH589 (Panobinostat) was a gift from Novartis Pharmaceuticals (Basel, tumors are unknown. Site-specific acetylation is an important Switzerland). Annexin V fluorescein isothiocyanate was obtained from BD regulatory modification that influences protein--protein interac- Biosciences (San Diego, CA, USA). Phospho specific antibodies for STAT3 tion. In solid tumor cancer cells, Yuan et al.,10 demonstrated that (Tyrosine 705), STAT3 (Serine 727), STAT1 (Tyrosine 701), STAT5 (Tyrosine STAT3 dimerization is regulated by reversible acetylation of lysine 694) and JAK2 (Tyrosine 1007/1008) were obtained from Cell Signaling at 685 in the SH2 domain of STAT3. Two classes of enzymes, Technology (Beverly, MA, USA). Phospho-tyrosine antibody was from histone acetyltransferases and histone deacetylases (HDACs), Millipore (Billerica, MA, USA). Antibodies for acetylated STAT3 (Lysine 685), reversibly regulate the extent of protein acetylation. In humans, STAT3, STAT1, STAT5, JAK2, JAK1, HDAC1, HDAC3, HDAC2, acetylated H3, HDACs are divided into three classes: class-I (HDAC1, HDAC2, acetylated H4, Bcl-2, Bcl-XL, Mcl-1, Bim, Bak, Bax, cMyc, cyclin D1 and p21 HDAC3 and HDAC8); the class-II (HDAC4, HDAC5, HDAC6, HDAC7, were all purchased from Cell Signaling Technology. Normal rabbit IgG

Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN, USA. Correspondence: Dr M Gupta or Dr TE Witzig, Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA. E-mail: [email protected] or [email protected] 1These authors contributed equally to this work. Received 23 May 2011; revised 3 October 2011; accepted 24 October 2011; published online 25 November 2011 Interplay between HDAC3 and STAT3 M Gupta et al 1357 control antibody was also from Cell Signaling Technology. Normal mouse streptavidin-conjugated Alexa fluorchrome (SA-AF594). Slides were IgG control antibody was purchased from Santa Cruz Biotechnology (Santa mounted with mounting media containing DAPI. Cruz, CA, USA). SHP1 and SHP2 antibodies were purchased from Cell Signaling Technology. Recombinant human IL-10 was purchased from R&D Cell-survival and -proliferation assay Systems (Minneapolis, MN, USA). 6 Cells (1 Â 10 /well) were cultured in the presence of various concentrations of LBH for 48 h or otherwise indicated and flow-cytometry assay was Lymphoma samples performed as previously described.18 Cell proliferation was determined by Tumor samples of DLBCL were obtained through the University of Iowa/ the tritiated-thymidine method.18 Mayo Lymphoma SPORE Biospecimens Core and the Predolin Foundation Biobank. Patients signed informed consent to provide excess tissue for Western-blotting and co-immunoprecipitation assay research on a protocol approved by the Mayo Clinic Institutional Review 5 Â 106 cells were incubated with various concentrations of LBH at 37 1C Board. A total of 12 primary patient samples of DLBCL were used for this 18 study; in 6 of these patients cryopreserved tumor cell suspensions were for various lengths of time, as indicated. To the lysates, 2--5 mg of specific available. All 12 DLBCL cases were sub-grouped into GCB or ABC subtype antibodies were added and complexes were allowed to form by incubating 1 based on the Hans immunohistochemistry algorithm applied to paraffin- with rotation at 4 C. A 50% slurry (25 ml) of protein A-Sepharose or protein embedded tumor samples.17 CD19 þ B-cells from the peripheral blood G-Sepharose was then added and the incubation continued for 2 h. from normal healthy blood donors were used as normal B-cell controls. The Immunoprecipitates captured with Sepharose beads were washed four SUDHL-6 (DHL6), OCI-Ly7 (Ly7), OCI-Ly19 (Ly19), OCI-Ly1 (Ly1), OCI-Ly3 times with RIPA buffer and analyzed by immunoblotting. (Ly3), SUDHL-2 (DHL2), U2932 and HBL1 DLBCL cell lines were a kind gift from Dr Louis Staudt. All cell lines were cultured in Iscove’s modified Transfection Dulbecco’s medium supplemented with 20% human serum. DLBCL lines For small interfering RNA (siRNA) experiments, cells were transfected with were serum starved with 0.5% bovine serum albumin overnight and then human B cell Nucleofector kit (Amaxa Biosystems Lonza, Gaithersrburg, treated with rIL-10. MD, USA). Briefly, 10 Â 106 cells were transiently transfected with 250 or 500 nM of HDAC3 siRNA (Santa Cruz Biotechnology) using the U-15 6 Immunohistochemistry and Immunoflorescence program. For plasmid transfection, 10 Â 10 cells were transfected with HDAC3 or HDAC1 plasmid by electroporation. HDAC3 and HDAC1 plasmids Sections (4-mm thick) were cut from formalin-fixed paraffin-embedded were provided by Dr Hungwen Chen (National Taiwan University). tissue blocks and stained as previously described with antibodies to HDAC3, HDAC1 and pSTAT3.18 For immunoflorescence microscopy, cytospins were prepared from LBH589-treated and -untreated cells, dried Nuclear and cytoplasmic analysis and fixed with 10% neutral-buffered formalin. Cells were permeabilized Nuclear and cytoplasmic extracts were separated with NE-PER nuclear and with 0.25% Triton X and antibody to STAT3 was added to respective slides. cytoplasmic extraction kit from Thermo Scientific (Kalamazoo, MI, USA) as Biotinylated anti-rabbit secondary antibody was added followed by instructed.

Figure 1. Expression of HDACs and pSTAT3 in ABC and GCB diffuse large B-cell lymphoma: (a) Expression of HDAC1, HDAC3 and pSTAT3Tyr705 were assessed in ABC and GCB patient samples by immuno-histochemistry. A representative example (ABC ¼ 7; GCB ¼ 5) is shown (400 Â ). (b, c) Expression of HDAC1, 2, 3, pSTAT3 Tyr705 and STAT3 were assessed at protein level by immunoblotting in ABC and GCB lymphoma cells from patients (b); in ABC and GCB human cell lines (c). (d) Expression of HDAC1, 2 and 3 in CD19 þ B(n ¼ 3) cells by immunoblotting.

& 2012 Macmillan Publishers Limited Leukemia (2012) 1356 --1364 Interplay between HDAC3 and STAT3 M Gupta et al 1358 RESULTS repeated in DLBCL cell lines and produced similar results. The Co-expression of class-I HDAC and pSTAT3Tyr705 in subtypes of ABC-type DLBCL cell lines Ly3 and Ly10 showed higher HDAC3 DLBCL tumors expression than the GCB lines Ly7 and DHL-6 (Figure 1c). Both Increasing evidence suggests that the regulation of gene types expressed HDAC1 with slightly higher levels in ABC; both expression by STATs requires class-I HDAC activity.19 DLBCL ABC and GCB cells expressed HDAC2 to a comparable level Tyr705 human samples typed as ABC and GCB by immunohistochemistry (Figure 1c). The ABC lines showed positivity for pSTAT3 and were stained for tyrosine pSTAT3 and HDAC class-I (HDAC1 and expressed higher total STAT3 compared with GCB cells. Normal HDAC3) expression. Both ABC and GCB tumors were HDAC1 peripheral blood CD19 þ B cells expressed HDACs 1 and 2, but not HDAC3 (Figure 1d). These results suggest that HDAC3 is positive (Figure 1a). All ﬁve ABC tumors were HDAC3 positive Tyr705 compared with only one of ﬁve GCB tumors (Figure 1a). aberrantly co-expressed with pSTAT3 in ABC DLBCL cells but Furthermore, all ABC tumors showed very strong nuclear positivity not in normal B-cells or GCB DLBCL. for phosphorylated STAT3 at tyrosine 705 residue; GCB cells were negative or only weakly positive (Figure 1a). On immunoblotting, HDAC3 accumulates in the nucleus and forms complex with STAT3 all three ABC DLBCLs tested expressed HDAC3 protein (Figure 1b), Nuclear and cytoplasmic analysis of pSTAT3Tyr705-positive Ly3 cell whereas only 1/3 GCB samples expressed HDAC3. HDAC1 line showed that HDAC3 predominantly resides in the nucleus expression was observed in four of six cases (2/3 ABC and 2/3 (Figure 2a). STAT3 was found in both cytoplasm and nucleus; GCB). All cases expressed total STAT3, with higher amounts in whereas activated STAT3 Tyr705 was restricted to the nucleus. ABC-type DLBCL. pSTAT3Tyr705 was only found in ABC-type DLBCL We investigated whether STAT3 and HDAC3 could physi- patient samples (3/3) (Figure 1b). The above studies were cally interact. First, we pulled down endogenous HDAC3 from

Figure 2. Interaction between STAT3 and HDAC3 in subtypes of DLBCLs: (a) Nuclear and cytoplasmic distribution of HDAC3/HDAC1 and STAT3 (both phosphorylated and unphosphorylated) in Ly3 cells. (b) HDAC3 was immunoprecipitated from lysates of two ABC and two GCB cell lines and the immuno-complex was examined for STAT3. (c) The immunoprecipitation control with normal rabbit IgG and beads. (d) HDAC3 was immunoprecipitated from two STAT3 þ and one STAT3-DLBCL patient cells and immuno-complex were blotted for STAT3. (e) STAT3 was pulled down from two ABC cell lines (Ly3 and DHL2) and blotted with HDAC1, HDAC3 and STAT3 antibodies. (f) The immunoprecipitation control with beads alone.

Leukemia (2012) 1356 --1364 & 2012 Macmillan Publishers Limited Interplay between HDAC3 and STAT3 M Gupta et al 1359 whole-cell lysates from two pSTAT3Tyr705-positive (Ly3, DHL2) and (Figure 3a) and late time points (Figure 3b). Immunoprecipita- two pSTAT3Tyr705-negative (Ly19, DHL6) DLBCL lines and blotted tion with control IgG failed to demonstrate these complexes with a specific STAT3 antibody. Precipitation of HDAC3 revealed a (Figure 3c). Even low doses of LBH (10 nM) were able to increase strongassociationwithSTAT3inboth DHL2 and Ly3 cells (Figure 2b). STAT3 acetylation. We have also seen some increase in STAT3 We did not observe any association of HDAC3 with STAT3 in either acetylation in response to LBH treatment in whole cell lysates Ly19 or DHL6 (pSTAT3-negative) cell lines (Figure 2b). Whole-cell (Figure 3a--lower panel). Similar results were obtained when total lysates (bottom panel Figure 2b) showed that both Ly19 and DHL6 STAT3 was pulled down from Ly3 and DHL2 cells and probed with express low amounts of STAT3 and HDAC3. These complexes were acetyl lysine antibody (Figure 3d). CREB-binding protein and its not detectable in the immunocomplexes obtained using a control homologue p300 is a transcriptional co-activator that regulates IgG antibody or beads alone (Figure 2c). We tested whether this STAT3 activity.21 Wang et al.22 have shown that STAT3 is association also is found in pSTAT3Tyr705-positive DLBCL patient acetylated by p300 both in vitro and in vivo. To demonstrate that samples. Indeed, in both pSTAT3-positive DLBCL tumors HDAC3 p300 is complexed with STAT3 in lymphoma cells, we pulled down and STAT3 were complexed (Figure 2d). endogenous p300 with a p300-specific antibody and probed for Furthermore, STAT3 was found to be associated with HDAC3 STAT3. p300 and STAT3 were shown to physically interact and suggesting a reciprocal relationship between these two proteins form complexes in both pSTAT3 þ DLBCL cell lines (Figure 3e) (Figure 2e). These complexes were not detected in precipitates compared with IgG control (Figure 3f). Further experiments were using beads alone (Figure 2f). performed to learn if STAT3 in DLBCL cells could be acetylated by p300 and if the level of acetylation at lysine 685 could be HDAC inhibition with LBH acetylates STAT3 at Lys685 increased with the HDAC inhibitor LBH. As indicated in Figure 3g, a As acetylation of STAT3 alters the distribution rather than the low level of acetylated STAT3 was found in the p300 immuno- functional status of STAT3,20 we next examined the effect of HDAC precipitate from untreated cells that increased with LBH treatment inhibition with LBH on endogenous STAT3 acetylation and in both Ly3 and DHL2 cells. localization of STAT3 in pSTAT3Tyr705-positive DLBCL cells. STAT3 was immunoprecipitated from Ly3 whole-cell extracts and blotted HDAC activity is required for cytokine-stimulated STAT3 nuclear for acetylated STAT3 with acetyl lysine antibody. The basal translocation acetylation level of endogenous STAT3 was low but increased We next examined by immunoblot and immunofluorescence after LBH treatment in a dose-dependent manner in early microscopy whether HDAC inhibition altered the nuclear versus

Figure 3. Effect of HDAC inhibition on STAT3 acetylation: (a, b) Ly3 cells were treated with various concentrations of LBH for 8 h (a) and 24 h (b) and then lysed. Lysates were immunoprecipitated with STAT3 antibody followed by western blot using STAT3Lys685 antibody. (c) Mouse IgG was used as control antibody for STAT3 immunoprecipitation. (d) Ly3 and DHL2 cells were treated with LBH for 8 h, lysed and immunoprecipitated with STAT3 antibody. Blot was then probed with acetyl lysine antibody. (e) Lysate from two ABC and two GCB lines were pulled down with p300 antibody and blotted with STAT3 and p300 antibodies. (f) Rabbit IgG was used as control for p300 immunoprecipitation. (g) Ly3 and DHL2 cells were treated with LBH lysed and then immunoprecipitated with p300 and examined for acetylated STAT3.

& 2012 Macmillan Publishers Limited Leukemia (2012) 1356 --1364 Interplay between HDAC3 and STAT3 M Gupta et al 1360 HDAC inhibition specifically abolishes tyrosine phosphorylation of STAT3 STAT3 is modified in vivo at amino- and carboxy-terminal sites by posttranslational modifications, including phosphorylation and acetylation.23 To determine whether HDAC inhibition leads to impaired activation of STAT3, we selected two pSTAT3Tyr705 DLBCL lines (DHL-2 and Ly3), along with pSTAT3-positive DLBCL patient samples. The samples were treated with LBH and the effect on activated and total STAT3 was examined. LBH was able to dephosphorylate STAT3 tyrosine phosphorylation in a dose- dependent manner in Ly3 cell within 24 h (Figure 5a). LBH had very little inhibitory effect on pSTAT3 serine and total STAT3 levels (Figure 5a). We have seen similar results when pSTAT3 DLBCL patient samples were treated with LBH (Figure 5b). We also investigated the effect of LBH using short-term (8 h) treatment in Ly3 and DHL2 cells. LBH caused a dose-dependent decrease in phosphorylation of STAT3 protein at tyrosine residue without much effect on pSTAT3 serine levels (Figure 5c). The effect of LBH on the phosphorylation of STAT1 and STAT5 was unable to be evaluated because they were not constitutively activated in Ly3 cells (Figure 5d). As JAK2 is a known upstream activator of STAT3 signaling, we investigated the effect of LBH on JAK2 phosphorylation by western blotting. LBH was not able to inhibit JAK2 phosphorylation in the Ly3 and DHL2 cell lines (data not shown). Collectively, these results demonstrate that LBH is specifically targeting STAT3 signaling in DLBCL tumors that express pSTAT3. To further confirm the effect of LBH on JAK/STAT signaling, we determined the effect of LBH on tyrosine activity of STAT3 and its upstream targets JAK2 and JAK1 (Figures 5e--h). We immunoprecipitated endogenous STAT3, JAK2 and JAK1 with antibodies to STAT3, JAK2, JAK1 and normal IgG control and blotted with phospho-tyrosine antibody. Figure 5e showed that LBH treatment results in significant reduction of the phosphotyrosine levels of STAT3. Immunoprecipitation with control IgG did not show baseline STAT3 tyrosine activity (Figure 5f). However, LBH treatment did not suppress phosphotyrosine levels of JAK2 and Figure 4. Effects of HDAC inhibition on STAT3 nuclear and cytoplasmic localization: (a) Serum-starved Ly3 cells were pretreated JAK1 (Figures 5g--h). These data suggest that STAT3-upstream with LBH for 60 and 240 min and then treated with IL-10 (100 ng/ml) pathway components are not targets of deacetylase activity. SHP1 for 30 min. Nuclear and cytoplasmic extracts were separated and and SHP2 are known to be involved in the dephosphorylation of blotted for antibodies as indicated. (b) Localization of STAT3 was STAT3 in keratinocytes.24 Next, we determined that whether HDAC examined by immunoflorescence in LBH-treated Ly3 cells as inhibition through LBH dephosphorylate STAT3 by activating described in materials and methods section. STAT3-specific phosphatases, such as SHP1 and SHP2. Our data in Ly3 cells demonstrate that LBH was not able to activate SHP1 and cytoplasmic localization of STAT3. Immunoblot analysis showed SHP2 protein expression (Supplementary Figure S2) indicating that in serum-starved cells STAT3 was distributed in both the that these phosphatases are not involved in LBH-mediated STAT3 cytosol and nucleus (Figure 4). A time-course analysis of IL-10 dephosphorylation. (a known inducer of the STAT3 pathway) in serum-starved Ly3 cells resulted in an increase in both the nuclear and cytoplasmic content of pSTAT3 (Supplementary Figure S1). However, IL-10 was pSTAT3-positive DLBCLs are more sensitive to HDAC inhibition able to increase Ac STAT3 predominantly in cytoplasm. Acetylated than pSTAT3-negative DLBCLs STAT3 was upregulated as early as 15 min with IL-10 treatment We investigated the effect of LBH on cell viability in pSTAT3- and the level was sustained up to 2 h (Supplementary Figure S1). positive DLBCL lines- Ly3, HBL1 and U2932 and the pSTAT3- However, phoshorylated-STAT3 levels were sustained up to 30 min negative DLBCL lines- DHL-6, Ly1 and Ly19. As shown in Figures and tended to decline over time (Supplementary Figure S1). 6a--c, significant inhibition of survival was observed in all three Pretreatment with LBH for 240 min blocked IL-10-induced pSTAT3 þ DLBCL cell lines with LD90 values in the 25-nM range. (30-min treatment) STAT3 nuclear translocation with a slight In contrast, the pSTAT3- lines DHL6, Ly1 and Ly19 cells were less increase in cytosolic STAT3 (Figure 4). Although, there was no sensitive to LBH-induced inhibition of survival at the indicated pSTAT3Tyr705 in either the cytosol or nuclear compartment at doses under the same experimental conditions (Figures 6d--f). baseline, IL-10 induced pSTAT3Tyr705 within 30 min in the cytosol To further investigate the nature of pSTAT3-positive DLBCL cell and nucleus. LBH was able to inhibit nuclear pSTAT3Tyr705 at 240 min, death induced by LBH, we measured the levels of PARP cleavage with little effect on cytosolic pSTAT3 (Figure 4). Interestingly, IL-10 in Ly3 cell lines, following LBH treatment. As shown in Figure 6g, was able to induce STAT3 acetylation at lysine 685 only in the LBH treatment induced PARP cleavage in Ly3 cells at low cytoplasm, which was slightly increased by LBH addition. concentrations (10 nM). We have also seen that in Ly3 cells, Ly3 cells endogenously expressing STAT3 were treated with the basal level of acetylated-histones H3 and H4 was low but LBH and immuno-stained with an STAT3 antibody. At baseline, Ly3 increased significantly in a dose-dependent manner with LBH cells showed STAT3 in both nuclear and cytoplasmic cell compart- treatment (Figure 6h). ments (Figure 4b); with LBH treatment, nuclear STAT3 staining was We next evaluated the effect of LBH on survival proteins abolished and STAT3 was found exclusively in the cytoplasm. downstream of STAT3. STAT3 has been previously shown to

Leukemia (2012) 1356 --1364 & 2012 Macmillan Publishers Limited Interplay between HDAC3 and STAT3 M Gupta et al 1361

Figure 5. Effects of HDAC inhibition on STAT3 signaling: (a) Ly3 cells were treated for 24 h with various concentrations of LBH. Whole-cell lysates were then subjected to SDS--PAGE followed by immunoblotting using pSTAT3 Tyr705 and pSTAT3 Ser727 antibodies. (b) Cells from ABC and GCB patients were treated with LBH for 8 h and blotted for pSTAT3 Tyr705 antibody. (c) Ly3 and DHL2 cells were treated with various concentrations of LBH for 8 h and and then blotted with pSTAT3 Tyr705 and pSTAT3 Ser727 antibodies. (d) Ly3 cells were treated with various concentrations of LBH and examined for pSTAT1Tyr701 and pSTAT5Tyr694 levels. (e--h) After a 24-h treatment with LBH, STAT3 immunoprecipitates (e), JAK2 immunoprecipitates (g) and JAK1 immunoprecipitates (h) were probed with phospho-tyrosine antibody. (f) Immunoprecipitation control with mouse IgG antibody is shown.

regulate Bcl-2 family anti-apoptotic protein expression, including HDAC3 has a role in STAT3 acetylation/deacetylation Bcl-xL, Mcl-1 and Bcl-2.25,26 DLBCL cells that are pSTAT3 positive Type-I HDACs regulate transcription activation by STATs.28 To expressed higher levels of the anti-apoptotic protein Mcl-1 but determine whether HDAC3 contributes to STAT3 deacetylation, similar levels of Bcl-2 and Bcl-xL (Supplementary Figure S3). In we tested the effect of overexpression of HDAC3 on the pSTAT3 positive cells, treatment with LBH depleted the levels of acetylation state of endogenous STAT3 at Lys-685. Ly3 cells were the Mcl-1 compared with pSTAT3-negative cells. We observed no transfected with or without an increasing amount of HDAC3. The effect on Bcl-2 and Bcl-xL in either pSTAT3-positive or -negative transfected cells were lysed and immunoprecipitated with anti- cells (Figure 6i). Next, we evaluated the effect of LBH on pro- STAT3 antibody, and immunoblotting was performed with anti- apoptotic proteins BAK, BAX and Bim. When compared with AcSTAT3 Lys685 or anti-STAT3 antibody. Overexpression of HDAC3 untreated controls, exposure to LBH resulted in slight increase in resulted in a decreased acetylation state of STAT3 (Figure 7a). In a the expression of Bim, particularly BimEL in Ly3 cells, however, no similar experiment, HDAC1 over-expression did not have much changes were noted in the expression of BAX and BAK in both effect on STAT3 acetylation (Figure 7b). Having established that pSTAT3-positive versus pSTAT3-negative DLBCL cells (Supplemen- HDAC3 physically associates with STAT3 and that LBH treatment tary Figure S3). increases STAT3 acetylation in ABC DLBCL cells, we then assessed Both cyclin D1 and c-Myc are STAT3 transcriptional targets and the ability of HDAC3 to deacetylate STAT3. For this, we employed promote cell-cycle progression. LBH inhibited c-Myc expression in siRNA to deplete endogenous HDAC3 in Ly3 cells. As shown in both pSTAT3-positive and -negative DLBCL cells; however, no Figure 7c HDAC3 knockdown upregulated STAT3 acetylation, detectable effect was observed on cyclin D1 expression (Figure 6j). indicating that HDAC3 negatively regulates the STAT3 acetylation HDACi have been shown to induce p21 expression.27 LBH in vivo. HDAC3 inhibition also inhibits constitutive STAT3 tyrosine treatment was able to induce p21 in a dose-dependent manner phosphorylation without effect on total STAT3 (Figure 7c). in both pSTAT3-positive and -negative DLBCL cells (Figure 6j). LBH HDAC class-1 histone (speciﬁcally, HDAC3) is required for cell effects on the transcriptional regulation of Mcl-1 were then growth and is involved in the apoptotic process via the regulation examined using reverse transcriptase-PCR analysis. LBH resulted in of pro-apoptotic genes.29,30 To evaluate whether this was true for moderate transcriptional inhibition of Mcl-1 gene expression at malignant lymphoma, we treated Ly3 cells with either HDAC3 or 24 h (data not shown). control siRNA for 96 h. HDAC3 transfected cells at 500 nM showed

& 2012 Macmillan Publishers Limited Leukemia (2012) 1356 --1364 Interplay between HDAC3 and STAT3 M Gupta et al 1362

Figure 6. Differential effects of LBH on pSTAT3-positive versus pSTAT3-negative DLBCL survival: (a--e) pSTAT3-positive DLBCL lines Ly3, HBL1 and U2932 (a--c) and pSTAT3-negative DLBCL lines DHL6, Ly1 and Ly19 (d--f) were treated with various concentrations of LBH for 48 h and survival was assessed by ﬂow cytometry. Data represent the mean±s.d. of three independent experiments. (g, h) pSTAT3 þ (Ly3) and pSTAT3À (DHL6) cells were treated with various concentrations of LBH and lysates were probed for PARP (g) and acetylated H3 and H4 (h) antibodies. (i, j) pSTAT3 þ (Ly3) and pSTAT3À (DHL6) cells were treated with LBH for 24 h and lysates were probed with indicated antibodies.

Figure 7. Effects of HDAC3 overexpression and inhibition on STAT3 acetylation, phosphorylation and survival: (a, b) Forty-eight hours after transfection with HDAC3 (a) and HDAC1 (b) plasmid, Ly3 cells were harvested and subjected to immunoprecipitation with STAT3 antibody. Blots were then examined for acetylated STAT3 and STAT3. (c) Forty-eight hours after siRNA transfection targeting HDAC3; Ly3 cell were harvested and lysed and immunoblotted for antibodies indicated. (d) Effect on HDAC3 inhibition through HDAC3 siRNA was assessed on Ly3 cell survival.

Leukemia (2012) 1356 --1364 & 2012 Macmillan Publishers Limited Interplay between HDAC3 and STAT3 M Gupta et al 1363 a 50% reduction in survival as detected by Annexin /PI staining pSTAT5 are not present in these cells and LBH did not inhibit compared with control (Figure 7d). pJAK1 and pJAK2 levels. The mechanism(s) underlying HDAC- mediated regulation of STAT3 tyrosine phosphorylation and acetylation remains elusive. It has been shown that acetylated DISCUSSION lysine 685 is situated on the SH2 domain of STAT3 where tyrosine DLBCL tumors can have different molecular signatures that can residue 705 is located.23 These two posttranslational modifications predict survival. 1 Delineating which oncogenic signaling path- may work mutually. One possibility is that LBH-induced STAT3 ways are implicated in DLBCL sensitivity and resistance are lysine acetylation prevents its tyrosine phosphorylation, thereby important to the development of new therapeutic strategies. In shutting down STAT3 activation. A recent study showed that order to improve outcome in patients with ABC DLBCL, we STAT1 acetylation counteracts IFN-induced STAT1 phosphoryla- explored how these tumors are regulated from a signaling tion,38 supporting the hypothesis that STAT3 acetylation regulates perspective. Recently, Ding et al.8 showed that STAT3 is its phosphorylation. Both phosphorylation and acetylation are constitutively activated in the ABC group of DLBCL. Constitutive modulators of protein functions and interactions and obviously STAT3 activation may result from the expression of upstream are a large potential area of research. By using STAT3K685R tyrosine kinases or from autocrine stimulation by cytokines or mutants, Yuan et al.10 showed that STAT3 lys685 acetylation is growth factors. These mechanisms may not be generally sufficient critical for STAT3 dimers required for cytokine-stimulated tran- for lymphomagenesis but may be important for the growth and scriptional regulation. Additional studies are required to elucidate resistance of DLBCL. The present study uncovered a regulatory the mechanisms by which HDAC3 regulate the activation of STAT3 pathway of STAT3 in ABC DLBCL that has implications for therapy. in ABC DLBCL. The effects of HDACi on gene expression are In leukemia, regulation by HDACs can result in aberrant gene thought to be highly selective and lead to transcriptional transcription.31 We have shown in this study that HDAC3 is not activation of genes, including the cyclin-dependent kinase expressed in normal blood B --cells, but is highly expressed in inhibitor p21. They also repress other genes important in control DLBCL tumors that have high levels of STAT3 (total and of cell growth. Our results clearly show that LBH can inhibit the phosphorylated). However, we did find one patient sample out STAT3-regulated anti-apoptotic protein Mcl-1 without much effect of five studied that was GCB and HDAC3 þ that did not express on Bcl-2 and Bcl-XL in ABC cells. However, the effect of LBH on pSTAT3 indicating that other factors can interfere with the cell-cycle proteins p21, cMyc and cyclin D1 was found to be very HDAC3/STAT3 interaction and hence block STAT3 signaling. similar in pSTAT3-positive and -negative cells. Taken together, it Further studies in larger numbers of patient samples of DLBCL appears that Mcl-1 overexpression through STAT3 is a mechanism are required to understand the frequency of this abnormality and of resistance in pSTAT3 DLBCL cells. the clinical significance. These results have therapeutic importance for DLBCL. Our data HDACs typically exist as components of large, multi-subunit show that LBH has potent activity in DLBCL, especially when the complexes that interact with other cellular proteins.32 Indeed, we DLBCL cells are dependent on STAT3 for survival. We demonstrate found that HDAC3 and pSTAT3 are primarily located in the that HDACi anti-apoptotic activity is not dependent on primary nucleus; whereas unphosphorylated STAT3 was found in both histone targets but rather on targeting the non-histone protein compartments. HDACs can function as enzymes to deacetylate STAT3 and ultimately constitutive STAT3 signaling. Our data also histones and regulate transcription. Although histones represent a clearly shows that HDAC3 is the important HDAC to be targeted in primary target for the physiological function of HDACs, the DLBCL. This suggests the potential to select patients for LBH antitumor effect of HDAC inhibitors might also work by therapy that have tumors that overexpress pSTAT3. Currently, a modulating the acetylation status of a series of non-histone number of HDAC inhibitors are in clinical trials for the treatment of proteins.33,34 In our studies, treatment of pSTAT3 DLBCL cells with various malignancies. Specifically, LBH589 is in clinical trials for the the HDAC inhibitor LBH increased the level of acetylated STAT3 at treatment of lymphoma and multiple myeloma.39,40 lysine 685. Histone acetyltransferase CBP/p300 is an important transcriptional coactivator capable of regulating acetylation of a wide range of transcription factors, including p53, E2F, AP-1 and CONFLICT OF INTEREST 35,36 NF-kB. Our data show that p300 and STAT3 physically The authors declare no conflict of interest. associate with each other in ABC cells (and to a lesser extent in GCB cells). Our pull-down assay further demonstrated that LBH also increases the p300-mediated acetylation of STAT3. After ACKNOWLEDGEMENTS ligand stimulation, latent STATs in the cytoplasm shuttle to and This work is supported by Lymphoma SPORE (P50 CA097274) Career Development accumulate in the nucleus by crossing through nuclear pore Award to MG (from NCI/ University of Iowa/Mayo Clinic); R01CA127433 to TEW and complexes.37 STAT3 predominately resides in the cytoplasm of the Predolin Foundation. unstimulated cells; upon stimulation, it quickly translocates into the nucleus and binds to the DNA of target genes, inducing their expression.18 Treating cells with HDACi LBH increases STAT3 AUTHOR CONTRIBUTIONS acetylation and results in decreased STAT3 protein in the nucleus MG and TEW designed the research. MG interpreted and analyzed all the data, along with a significant decrease in nuclear phosph-STAT3 at made the figures and wrote the manuscript. MG, MJS, JJH and LEW performed the tyrosine. This suggests that HDAC inhibition may increase STAT3 experiments. 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