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. Author manuscript; available in PMC: 2011 Oct 9.
Published in final edited form as: Br J Haematol. 2010 Sep 29;151(4):387–396. doi: 10.1111/j.1365-2141.2010.08342.x

The class-I HDAC inhibitor MGCD0103 induces apoptosis in Hodgkin lymphoma cell lines and synergizes with proteasome inhibitors by an HDAC6-independent mechanism

Daniela Buglio 1, Vidya Mamidipudi 2, Noor M Khaskhely 1, Helen Brady 2, Carla Heise 2, Jeffrey Besterman 3, Robert E Martell 3, Kyle MacBeth 2, Anas Younes 1
PMCID: PMC3189488  NIHMSID: NIHMS320181  PMID: 20880107

Summary

Inhibition of histone deacetylase 6 (HDAC6)-dependent aggresome function by pan HDAC inhibitors was recently reported to be a key mechanism underlying the synergistic activity between proteasome inhibitors and HDAC inhibitors in a variety of tumour types. Because these combinations induce significant thrombocytopenia in vivo, we examined whether less toxic, isotype-selective HDAC inhibitors may still synergize with proteasome inhibitors, and if so, by what mechanisms. Here, we showed that the class I HDAC inhibitor, MGCD0103, has a potent antiproliferative activity in Hodgkin lymphoma (HL) cell lines. Furthermore, MGCD0103 induced tumour necrosis factor α (TNF-α) expression and secretion, which was associated with nuclear factor (NF)-κB activation. Selective inhibition of TNF- α expression by short interfering mRNA, or inhibition of MGCD0103-induced NF-kB activation by proteasome inhibitors enhanced MGCD0103-induced cell death. Thus, our results demonstrate that MGCD0103 may synergize with proteasome inhibitors by HDAC6-independent mechanisms, providing mechanistic rationale for exploring this potentially less toxic combination for the treatment of lymphoma.

Keywords: Hodgkin lymphoma, new drugs for lymphoma, MGCD0103, Bortezomib

Histone deacetylases (HDACs) are promising targets for cancer therapy (Lane & Chabner, 2009; Marks & Xu, 2009). They are a family of enzymes that deacetylate lysine residues on histone and non-histone proteins, which play a role in regulating cell cycle progression and survival (Xu et al, 2007). The currently known 18 human HDACs are grouped into four classes; class I (HDAC 1, 2, 3 and 8), class II (HDAC 4, 5, 6, 7, 9 and 10), class III sirtuins (SIRT1-7), and class IV (HDAC 11). Pharmacological inhibitors are broadly classified as pan HDAC inhibitors that inhibit class I and class II enzymes, such as vorinostat and panobinostat; and class I inhibitors, such as MGCD0103 (Lane & Chabner, 2009). Although several HDAC inhibitors have demonstrated antiproliferative activity in vitro against a variety of tumour types, their clinical utility has been hampered by their in vivo toxic effects (Lane & Chabner, 2009). Furthermore, HDAC inhibitors frequently alter several survival and resistance pathways, they are explored as modulating agents in combination with a variety of anticancer drugs. For example, a synergistic effect was recently described between pan HDAC inhibitors and proteasome inhibitors. This synergy was attributed to the ability of pan HDAC inhibitors, such as panobinostat and vorinostat, to inhibit HDAC6-dependent aggresome function (Catley et al, 2006; Corcoran et al, 2004). However, in the clinical setting, both proteasome inhibitors and pan HDAC inhibitors induce significant thrombocytopenia, making this novel combination regimen rather toxic. In this study, we investigated whether class-I selective HDAC inhibitors, which have no significant haematological toxicity, may also synergize with proteasome inhibitors, and if so, by what mechanisms.

To answer these questions we evaluated the novel benza-mide-based HDAC inhibitor MGCD0103, which preferentially inhibits class I HDACs, especially HDAC1, with no effect on HDAC6 (Fournel et al, 2008; Gloghini et al, 2009). We and others have recently evaluated the single agent activity of MGCD0103 in patients with relapsed cancer, including Hodgkin lymphoma (HL), and confirmed its promising clinical activity and its lack of platelet toxicity (Younes et al, 2007). Here, we showed that MGCD0103 upregulated the cell cycle regulatory protein p21 and activates the intrinsic caspase pathway to induce apoptosis. Furthermore, MGCD0103 up-regulated the expression of several inflammatory cytokines, including tumour necrosis factor α (TNF-α), which was associated with nuclear factor (NF)-κB activation, attenuating MGCD0103 anti-tumour activity. Inhibition of TNF-α expression by short interfering RNA, or inhibition of MGCD0103-induced NF-κB activation by proteasome inhibitors enhanced MGCD0103 killing effect. Collectively, our data demonstrate that HDAC6 inhibition is not required for enhancing proteasome inhibitor activity in HL, providing additional mechanistic rationale for the development of potentially less toxic combination regimens of the class-I HDAC inhibitors and proteasome inhibitors for the treatment of cancer.

Materials and methods

Cell lines, cell culture, and reagents

The human Hodgkin and Reed–Sternberg (HRS)-derived cell lines were obtained from the German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany. The phenotypes and genotypes of these cell lines have been previously published (HD-LM2 of T cell origin, L428 and KMH2 of B cell origin), and all cell lines were not infected with the Epstein-Bar virus (Drexler, 1993). Cell lines were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), 1% l-glutamine, and penicillin/streptomycin in a humid environment of 5% CO2 at 37°C. The HDAC inhibitor suberoylanilide hydroxamic (vorinostat, SAHA) was purchased from Biovision, Inc. (Mountain View, CA, USA). MGCD0103 was kindly provided by Methylgene (Toronto, Canada). The proteasome inhibitor, Bortezomib (PS-341) was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA). Antibodies to TNF-α, NFkB p65, IKbα, phospho-IKbα, p21, p15, acetylated histone 3, caspase 3, 8, 9 and PARP [Poly (ADP-ribose) polymerase], were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for A20 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to β-actin was from Sigma Chemicals Co. (St. Louis, MO, USA). Antibodies to CD19, CD20, CD30, CD40, CD80, TRAIL-R1 and TRAIL-R2 were from BD Biosciences (San Jose, CA, USA).

HDAC enzyme assay in vitro

The effect of different HDAC inhibitors on the in vitro enzymatic activity of each HDAC isoform was performed by Reaction Biology Corporation (Malvern, PA, USA) (Hauser & Jung, 2009; Yuan et al, 2009). Briefly, full lengths of (human) HDAC1, HDAC2, HDAC5 and HDAC6 genes were expressed by baculovirus expression system in Sf9 cells, with GST tag in C or N-terminals. The full length human HDAC3 with C-terminal His tag and human NcoR2, N-terminal GST tag, were co-expressed as a complex in baculovirus expression system. The full length of human HDAC8 was expressed in an Escherichia coli expression system. The catalytic domains of human HDAC4 (AA 627- 1085 with N-terminal GST tag); human HDAC7 (AA 518-end with N-terminal GST tag), human HDAC9 (AA 604-1066 with C-terminal His tag) and human HDAC10 (AA 1-631 with N-terminal GST tag) were all expressed by baculovirus expression system in Sf9 cells. Enzymes were stored in 50 mmol/l Tris-HCl, pH 8.0, 138 mmol/l NaCl, 20 mmol/l glutathione, and 10% glycerol, and were stable for >6 months at −80°C, and the purity was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Peptide substrate, p53 residues 379-382 (RHKKAc), was conjugated with AMC (7-acetoxy-4-methyl coumarin). The free AMC was detected with excitation of 390 nm and emission 460 nm by using a fluorescent-based plate reader or microarray reader. Reaction Buffer was 25 mmol/l Tris/Cl, pH8.0, 137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 0.1 mg/ml bovine serum albumin (BSA). The HDAC reaction was performed using increasing concentrations of each compound (10−9 −10−3 mol/l) at 30°C for 2 h before adding the developer reagent. The free AMC was detected with excitation of 360 nm and emission 460 nm at kinetic mode for 90 min. The reaction slopes were then normalized and plotted with GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) to derive the IC50 values.

In vitro proliferation assay

Cells were cultured in 6, 12 and 24-well plates at a concentration of 0.5 × 106 cells/ml. Cell viability was assessed with the non-radioactive cell proliferation MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulf-ophenyl)-2H-tetrazolium) assay by using CellTiter96AQueous One Solution Reagent (Promega, Madison, WI, USA), as previously published (Georgakis et al, 2006). Briefly, 80 μl of cell suspension was incubated with 20 μl of CellTiter 96AQueous One Solution Reagent in 96-well plates for 1 h at 37°C, 5% CO2, and formazan absorbance was measured at 490 nm on a μQuant plate reader equipped with KC4 software (Biotek Instruments, Winooski, VT, USA). Each measurement was made in triplicate and the mean value was determined.

Flow cytometry

Cell surface expression was determined by fluorescence-activated cell sorting (FACS) as previously described (Zheng et al, 2003). Apoptosis was determined by Annexin-V-FLUOS and propidium iodide double staining (Roche Molecular Biochemicals, Indianapolis, IN, USA), according to the manufacturers' instructions and as previously published (Georgakis et al, 2006; Zheng et al, 2003). Cell cycle fractions were determined by propidium iodide nuclear staining. Briefly, cells were harvested, washed in phosphate-buffered saline, fixed with 70% ethanol, and incubated with propidium iodide for 30 min at 37°C. Data were collected on a FACSCalibur flow cytometer using Flowjo software (Tree Star Inc, Ashland, OR, USA), as described previously (Georgakis et al, 2006; Zheng et al, 2003). Results were obtained by analysing data with FlowJo software (Tree Star Inc.), and are shown as mean of three independent experiments.

Enzyme-linked immunosorbent assay (ELISA)

HL cell lines were incubated with 1 μmol/l of MGCD0103 or dimethyl sulphoxide (DMSO) for 24 h, before supernatants were collected and examined for TNF-α production by ELISA (R&D Systems, Minneapolis, MN, USA), according to the manufacturers' instructions and as previously published (Buglio et al, 2008). Each experiment was performed in triplicate and results represent mean value from three different experiments.

Western blot analysis

Total cellular proteins were extracted by incubation in lysis buffer (Cell Signaling Technology) for 30 min on ice and then centrifuged to remove cellular debris. The protein in the resulting supernatant was quantified by the bicinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL, USA) according to the manufacturer's instructions. Then, protein was diluted 1:2 in protein SDS loading buffer (Cell Signaling Technology), and heated to 95°C for 5 min. A total of 30 μg of protein was loaded onto 12% Tris-HCl SDS-PAGE Ready Gels (Bio-Rad, Hercules, CA, USA), transferred to a nitrocellulose transfer membrane (Bio-Rad), and detected by using Super-SignalWest Dura Extended Duration Substrate (Pierce Chemical Co.), as previously described (Georgakis et al, 2006; Zheng et al, 2003).

Real-time polymerase chain reaction (PCR)

Total RNA was extracted with the Qiagen (Valencia, CA, USA) RNeasy mini protocol and was converted to cDNA using oligo-dT, random hexamers, and iScript (Bio-Rad). After diluting cDNA in dH20 1:20, real-time PCR was performed using a sequence detector (model Bio-Rad iCycler). RT-PCR was performed on duplicate 1-μl cDNA samples using an Bio-Ra iCycler iQ5 sequence detector (Bio-Rad) and target mixes (JAK/STAT Signaling Pathway RT-PCR, SuperArray Bioscience Corporation, Frederick, MD, USA) with the following thermal cycling profile: 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 60 s. The assay used was: JAK/STAT Signaling Pathway RT-PCR. The expression of GAPDH was used as an internal standard in calculating relative gene expression. Results are represented as the mean three independent experiments.

Gene expression profiling

For these experiments, we focused our analysis on the two HL cell lines of B cell origin (L428 and KMH2). Cells were plated at 5 × 105 cells per 100 mm dish, incubated for 48 h, and then treated with MGCD0103 (0.02, 0.2 and 1 μmol/l) or SAHA (0.02, 0.2 and 1 μmol/l) for 24 h. Total RNA was isolated, and gene expression was evaluated using Affymetrix U133 Plus 2.0 chips. Results were analysed by GENESPRING 7.3 (Agilent; Santa Clara, CA, USA). After normalization, the ratios of gene expression of drug (MGCD0103 and vorinostat) treated to control cells (DMSO) from biological duplicate samples of 1.8-and more-fold regulated were defined as the differentially expressed genes. Pathway analysis was performed on this data using GeneGo MetaCore software (GeneGo, St Joseph, MI, USA).

Selective inhibition of TNFα expression by short interfering RNA (siRNA)

SiRNA oligonucleotides used to block TNF-α expression (RNA- UUA UCU CUC AGC UCC CCA UUG G) and nonspecific control siRNA (Primer #: 125793A07) were purchased from Invitrogen (Carlsbad, CA, USA). HL cell line (L428) was plated at a 1 × 106/ml concentration in 12-well plates. Double-stranded siRNAs (2 μmol/l) were transfected at time 0 h using using Nucleofection kit (Amaxa, Lonza Walkersville Inc., Walkersville, MD, USA) as previously published (Buglio et al, 2008). Cells were harvested after 24 h and were subjected to Western blot analysis. This protocol gave a transfection efficiency of between 60% and 70%.

Statistical methods, isobologram and combination index calculation

The effectiveness of the drugs and their combinations used in the present study were analysed by using the Calcusyn Software (Biosoft, Ferguson, MO, USA). The combination index and isobologram plot were calculated according to the Chou-Talalay method (Georgakis et al, 2006; Zheng et al, 2003). A combination index value of 1 indicates an additive effect between two drugs. Combination index values <1 indicate synergy, and the lower the value, the stronger the synergy. In contrast, combination index values >1 indicate antagonism. Effects of certain conditions on cell proliferation, apoptosis, and cytokine production were performed in three independent experiments in triplicate. The two-tailed Student t test was used to estimate statistical significance of the differences in results from the three experiments. The level of significance was set to 0.05.

Results

MGCD0103 induces apoptosis in HL cell lines

The in vitro specific inhibitory activity of MGCD0103, and two pan HDAC inhibitors (vorinostat, and Trichostatin A), were examined against purified HDAC 1-10 isoforms as described in the Materials and Methods. MGCD0103 preferentially inhibited HDAC1, with an IC50 of 154.5 nmol/l, but also inhibited the activity of HDAC2 and HDAC8. Consistent with its class-I selectivity, MGCD0103 had no effect of HDAC6 (Supplemental Fig S1). In comparison, vorinostat preferentially inhibited HDAC6 IC50 = 28 nmol/l), but also had activity against HDACs 1 (IC50 = 348.4 nmol/l), 3 and 8. Trichostatin-A, which was used as a positive control, demonstrated higher potency against HDACs 1-10 compared with MGCD0103 and vorinostat. The superiority of MGCD0103 over vorinostat in inhibiting HDAC1 activity in a cell-free assay, translated into a more potent antiproliferative activity in HL cell lines (Fig 1A). After 72 h of incubation, the IC50 for MGCD0103 in three HL cell lines ranged between 0.6 and 0.9 μmol/l compared with 1.8–2.8 μmol/l for vorinostat. At the molecular level, MGCD0103 acetylation of histone 3 and upregulation of the cell cycle regulatory protein p21 (Fig 1B) was observed with much lower concentrations compared with our previous experience with vorinostat (Buglio et al, 2008). Furthermore, MGCD0103 downregulated XIAP, activated caspases 9 and 3, and induced apoptosis (Fig 1C, D). After 48 h of incubation with 1 μmol/l of MGCD0103 or vorinostat, the percentage of apoptotic cells achieved was 59% vs. 21% respectively in the HD-LM2 cells; 72% vs. 15% in the L428 cells; and 69% vs. 13.8% in the KM-H2 cells (Fig 1D). Collectively, these data demonstrate that inhibition of class-I HDACs by MGCD0103 is sufficient to induce cell death in HL cell lines, suggesting that a more broad inhibition of HDAC enzymes, including HDAC6, is not required for an effective antiproliferative effect in vitro.

Fig 1.

Fig 1

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Antiproliferative effects of MGCD0103 in Hodgkin Lymphoma (HL) cell lines. (A) Cells were incubated with increasing doses (0.1-0.5-1-2-5 μmol/l) of MGCD0103 or vorinostat for 72 h before cell viability was determined by the MTS assay. MGCD0103 was more effective than vorinostat in all three HL cell lines, with a lower IC50 values as shown in the table. Results are mean of 3 independent experiments (±SEM). (B) Cells were incubated with 0.1–2 μmol/l of MGCD0103 for 24 h and whole cell lysates were examined for histone 3 acetylation (Ac-histone 3) and p21 using Western blot analysis. (C) MGCD0103 downregulated the level of the inhibitor of apoptosis protein XIAP, activated caspases 9 and 3, but had no effect on caspase 8. Vorinostat also downregulated XIAP, but was less potent in activating the caspase pathway. HL cell lines were incubated with medium or 1μM of MGCD0103 or vorinostat for 24 or 48 h. before cell lysates were examined by Western blot (D) A representative experiment of three independent experiments demonstrating a dose-response effect of MGCD0103 and vorinostat on induction of apoptosis in three HL cell lines as determined by propidium iodide (PI) and annexin-V staining and FACS analysis. Results are shown after 48 h of incubation. In all three of the cell lines, MGCD0103 was more potent in induction of apoptosis when compared with vorinostat.

MGCD0103 regulates the expression of the TNF superfamily and inflammatory cytokines

To better understand the mechanism of action of MGCD0103 in HL cell lines, we examined its effect on gene expression in the two cell lines that are of B-cell origin (L428 and KMH2). Cells were incubated with 0.02, 0.2, or 1 μmol/l of MGCD0103 or vorinostat for 24 h before gene expression profiling (GEP) was performed. This range of 3 different doses enabled us to examine the dose response effect of each drug on GEP, in addition to enabling the comparison of biologically equivalent doses of the two drugs on the same cell line (i.e. 0.2 μmol/l of MGCD0103 has similar antiproliferative effect to 1 μmol/l of vorinostat. A dose response effect on gene expression was observed in both cell lines and with both drugs. Lists of the MGCD0103 and vorinostat-regulated genes (>1.8-fold change) were analysed using NextBio in order to identify the affected biogroups from Gene Ontology Consortium (GO) (Fig S2). The main GO categories impacted by MGCD0103 involved the activation of immune or inflammatory responses against an external stimulus. We subsequently focused our analysis on the tumour necrosis factor superfamily (TNFSF) of ligands and receptors, and the JAK/STAT pathways, both of which are known to play key roles in regulating inflammation and survival in HL. MGCD0103 downregulated TNFRSF8 (CD30) receptor expression, a marker of the malignant Hodgkin and Reed-Sternberg cells (Figs 2A and S3). These results were further confirmed by RT-PCR and FACS analysis of CD30 surface protein expression (Fig S4). MGCD0103 significantly increased the expression of several TNFSF members that regulate inflammation and immunity, including TNFSF4 (OX40L), TNFSF9 (4-1BB) and TNF (Fig 2A) and upregulated the expression of genes that are involved in interferon gamma, IL6, IL8 and IL23 signaling pathways (Fig S5). Furthermore, MGCD0103 down-regulated the expression of the TH2 chemokine, Thymus and activation-regulated chemokine (CCL17) (Peh et al, 2001; Weihrauch et al, 2005). MGCD0103 also differentially regulated Jak/STAT signaling components, shifting the balance to favour cell death, including upregulation of Silencer of Cytokine Signaling 3 (SOCS3), STAT1 and STAT4, and downregulation of JAK2, JAK3, STAT5 and STAT6 (Fig 2B). Results from the GEP experiments on the JAK/STAT pathway were further confirmed using RT-PCR array (Fig 2C).

Fig 2.

Fig 2

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MGCD0103 regulationof TNF super family receptors and ligands and JAK/STAT pathway. (A) Gene expression profiling (GEP) of the KMH2 cell line was performed after incubation with increasing concentrations of MGCD0103 or vorinostat for 24 h. MGCD0103 significantly upregulated the expression of TNF, TNFSF4, TNFSF4 and TNFRSF15, but downregulated TNFRSF8. Similar results were observed inthe L428 cells (shownin Fig S3). (B) Effect of MGCD0103 on JAK/STAT signaling pathway. MGCD0103 significantly upregulated the expression of SOCS3, STAT4 and STAT1, but downregulated STAT5 (C) The effect of MGCD0103 on JAK/STAT pathway was confirmed by RT-PCR. The numbers shown in the table represent fold change in copy numbers of mRNA (mean of 2 independent experiments performed in triplicate). The level of expression of CDKN1A, which encodes for the cell cycle protein p21, is shown as a positive control. In addition to Jak/STAT molecules, the expression level of selected target genes is also shown.

MGCD0103 upregulates TNF-α and activates NF-kB

Because MGCD0103 upregulated the expression of TNF, we examined its effect on TNF-α cytokine secretion in HL cells supernatants. Using an ELISA assay, MGCD0103 profoundly increased TNF-α levels within 24 h of incubation (Fig 3A). The induction of TNF-α was associated with upregulation of NFKB1 gene expression (Fig 2A, C, phosphorylation of IkB-α, and the translocation of p65 NF-kB from the cytoplasm to the nucleus, indicative of NF-kB activation (Fig 3B). Next we examined the consequences of inhibiting TNF-α expression by siRNA (Fig 3C). We found that downregulation of endogenous TNF-α expression by siRNA had a minimal effect on HL cell survival (Fig 3D). In contrast, downregulating MGCD0103-induced TNF expression by siRNA potentiated MGCD0103 antiproliferative effect, suggesting that TNF-α may attenuate MGCD0103 activity in HL cells, perhaps by activating NF-kB (Fig 3D).

Fig 3.

Fig 3

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Effect of MGCD0103 on TNF-α secretion and NFk-B activation. (A) The effect of MGCD0103 on TNF expression was confirmed by detecting an increase in TNF-α protein levels in the supernatants of HL cell lines after treatment with MGCD103 (0.5 or 1 μmol/l) for 24 or 48 h. Each value represents the mean TNF-α measurement from three independent experiments performed in triplicate as determined by ELISA. (B) The increase in TNF-α secretion was associated with I-kB phosphorylation and NF-kB activation as indicated by the increase in p65 NF-kB level in the nuclear compartment. Western blot analysis was performed on subcellular nuclear fraction to examine p65 levels, or whole cell lysates to examine I-kB phosphorylation in response to MGCD0103 (1 μmol/l). (C) L428 cells were transfected with TNF-α or control siRNA, and after 48 h the cellular level of TNF-α was determined by Western blot. This is a representative of three independent experiments showing the efficacy TNF-α siRNA in downregulating TNF-α protein expression. (D) Downregulation of TNF-α decreased the viability of L428 cells and enahanced the killing effect of MGCD0103, as determined after 48 h of incubation by the MTS assay. Each value represents the mean of three independent experiments performed in triplicate (±SEM).

MGCD0103 synergizes with proteasome inhibitors

Recent studies demonstrated that pan HDAC inhibitors synergize with proteasome inhibitors by inhibiting HDAC6-mediated aggresome function (Corcoran et al, 2004). Because MGCD0103 has no inhibitory effect on HDAC6, we hypothesized that proteasome inhibitors may synergize with MGCD0103 by inhibiting NF-kB activation. To test this hypothesis, we examined the effect of the proteasome inhibitor bortezomib on MGCD0103-induced NF-kB activation. We found that bortezomib inhibited MGCD0103-induced NF-kB activation, as indicated by inhibiting p65 NF-kB nuclear transloaction (Fig 4A), which was associated with a synergistic antiproliferative effect, as determined by the annexin-V binding method (Fig 4B). The synergistic activity was also observed between MGCD0103 and another proteasome inhibitor NPI0052 (Fig 4C). Collectively, these data demonstrated that the class-I HDAC inhibitor MGCD0103 synergizes with proteasome inhibitors by HDAC6 independent mechanisms, by inhibiting MGCD0103-induced NF-kB activation.

Fig 4.

Fig 4

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Bortezomib synergizes with MGCD0103 by inhibiting its NF-kB activation. (A) HL cells were incubated with MGCD0103 (1 μmol/l), bortezomib (20 nmol/l), or both for 24 h before the nuclear fraction was examined for p65 NF-kB expression by Western blot. As shown, bortezomib inhibited MGCD0103-induced NF-kB activation. (B) Summary data from three independent experiments demonstrated a synergistic effect of MGCD0103 and bortezomib on induction of apoptosis in three HL cell lines as determined by propidium iodide (PI) and annexin-V staining and FACS analysis. Results are shown after 48 h of incubation. Each value is the mean of three independent experiments performed in triplicate (±SEM). *P value <0.05. (C) Synergy between MGCD0103 and bortezomib or NPI002 at different combination doses was determined by calculating the combination index analysed by CALCUSYN software. A combination index <1 indicates synergy.

Discussion

This study provided insights on the complex molecular mechanisms of MGCD0103 antiproliferative action in HL, and identified several pathways that are regulated by this class-I HDAC inhibitor. We found that MGCD0103 has a direct dose-dependent antiproliferative activity in HL cell lines, which was mediated by regulating a variety of cell cycle and survival proteins. Furthermore, our data suggest that MGCD0103 may indirectly inhibit tumour cell growth and survival by modulating key mediators of inflammation, immunity, and angiogenesis in the microenvironment. This hypothesis should be confirmed by performing carefully designed correlative pharmacodynamic studies on biospecimens obtained from patients enrolled on MGCD0103 therapy.

Interestingly, MGCD0103 downregulated TNFRSF8 (CD30 receptor) at the mRNA and protein levels. Whether CD30 receptor expression is required for HRS cell survival remains controversial. However, as several HDAC inhibitors and anti-CD30 monoclonal antibodies are being developed for the treatment of patients with HL, it will be important to examine the effect of CD30 downregulation on the activity of anti-CD30 antibodies.

Because MGCD0103 regulated the expression and repression of a large number of genes, it is inevitable that some of these genes had opposing functions. For example, MGCD0103 upregulated the expression of the gene encoding vascular endothelial growth inhibitor (TNFRSF15), a TNFSF member that inhibits angiogenesis, but also upregulated IL8 expression that may promote angiogenesis. Similarly, while MGCD0103 activated CASP9, downregulated XIAP and induced apoptosis, but also induced TNF and activated NFKB1, which attenuated MGCD0103-induced cell death. It is not clear how these conflicting signals would be manifested in the clinical setting. Future studies should investigate the contribution of each HDAC enzyme by selective knockdown experiments. Such studies may facilitate the development of even more isotype-selective HDAC inhibitors that are more potent but less toxic.

Although alteration in SOCS1 expression has been linked to the aberrant activation of Jak2, STAT5 and STAT6 in certain types of Hodgkin and non-Hodgkin lymphoma (Baus & Pfitzner, 2006; Mottok et al, 2007; Weniger et al, 2006). MGCD0103 had a modest effect on SOCS1 expression but, in contrast, it had a profound effect on SOCS3 expression, which may have played a role in downregulating STAT6 signaling. In fact, the complexity of HDAC inhibitors activity can be further illustrated by the effect on STAT6 phosphorylation. As shown in Fig 4, this effect may have been caused by a direct inhibition of STAT6 transcription, by inhibition of JAK2 transcription, and/or by upregulation of SOCS3, which may inhibit Jak2 function. Thus, the net effect of MGCD0103 was a result of a series of gene induction and repression, in addition to altering protein function, shifting the balance between different JAKSs and STATs to favour cell death and TH1-type immune response (Guo et al, 2006; Kim & Lee, 2007).

In addition to regulating the expression of inflammatory cytokines and chemokines, MGCD0103 regulated the expression of several mediators of innate and adaptive immunity, including IL8, CCL3 (MIP-1α), CXCL9, 10, and 11, TNFSF4, and TNFSF9 (Croft et al, 2009; Redmond & Weinberg, 2007; Vire et al, 2009; Wang et al, 2009). These mediators are known to attract and activate anti-tumour cellular immune responses involving granuloctyte, macrophage, and cytotoxic T cells. Understanding how HDAC inhibitors regulate the immune response could open the door for novel treatment approaches to cancer and autoimmune diseases (Croft et al, 2009; Redmond & Weinberg, 2007).

Previous studies demonstrated that HDAC6 inhibition was responsible for the synergistic activity between pan HDAC inhibitors and proteasome inhibitors, suggesting that class-I HDAC inhibitors may lose this potential synergistic advantage. In this study, we found that MGCD0103 upregulated the expression of several inflammatory cytokines, which in turn, activated NF-kB and attenuated its killing effect on tumour cells. Thus, inhibition of NF-kB activation by different proteasome inhibitors provides a mechanistic explanation of how proteasome inhibitors enhanced MGCD0103 activity, independent of HDAC6. In fact, several combination regimens of pan-HDAC inhibitors and proteasome inhibitors are being evaluated in clinical trials for the treatment of cancer. However, because both pan-HDAC inhibitors and proteasome inhibitors induce severe thrombocytopenia, these agents cannot be combined at full doses due to added platelet toxicity. In contrast, class-I HDAC inhibitors rarely induce thrombocytopenia, and therefore, are safer to combine with proteasome inhibitors.

In summary, this study sheds more light on the complex mechanisms of actions of class-I HDAC inhibitors in HL, providing a framework for the development of novel selective isotype-selective HDAC inhibitors for the treatment of lymphoma. Furthermore, our data provide a mechanistic rationale for combining class-I HDAC inhibitors with proteasome inhibitors for the treatment of selected cancers.

Supplementary Material

2

Fig S1. Effect of the class-I selective HDAC inhibitor MGCD0103 and two pan HDAC inhibitors (vorinostat and trichostatin-A on purified HDAC 1-10 enzymatic activity in cell-free assays.

Fig S2. Biogroups defined in NextBio are collections of genes sharing a specific biological function or pathway and could consist of signaling and metabolic pathways, protein families, gene ontologies, as well as other relevant gene sets.

Fig S3. GEP focusing on the TNFRSF pathway in the L428 cells.

Fig S4. (A) The effect of MGCD0103 on TNFRSF8 expression is confirmed by RT-PCR. (B) Summary results demonstrating the effect of MGCD0103 (1 μmol/l) on TNFRSF8 surface expression as determined by FACS analysis.

Fig S5. An example of the effect of MGCD0103 on inflammatory cytokine and chemokines using gene expression profiling (GEP) in the KM-H2 cell line.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgments

Anas Younes, M.D: This work was supported in part by NCI grant 1 R21 CA117070-01 and a lymphoma SPORE grant 1P50CA136411-01A1, Clay Chiles Lymphoma Fund, and Jack L. Stotsky Memorial Fund and by a grant from Pharmion and Methylgene.

Footnotes

Authorship contributions: D.B. performed the experiments, evaluated the results, and helped in writing the manuscript. V.M. performed the experiments, evaluated the results, and helped in writing the manuscript. N.M.K. performed the experiments, evaluated the results, and helped in writing the manuscript. R.E.M. provided important reagents for the studies, and approved the final manuscript. J.B. provided important reagents for the studies. C.H. designed the experiments, evaluated the results. K.M. designed the experiments, evaluated the results. A.Y. designed the experiments, evaluated the results, and approved the final manuscript. D.B. and V.M. contributed equally to this manuscript.

Supporting information Additional Supporting Information may be found in the online version of this article:

References

  1. Baus D, Pfitzner E. Specific function of STAT3, SOCS1, and SOCS3 in the regulation of proliferation and survival of classical Hodgkin lymphoma cells. International Journal of Cancer. 2006;118:1404–1413. doi: 10.1002/ijc.21539. [DOI] [PubMed] [Google Scholar]
  2. Buglio D, Georgakis GV, Hanabuchi S, Arima K, Khaskhely NM, Liu YJ, Younes A. Vorinostat inhibits STAT6-mediated TH2 cytokine and TARC production and induces cell death in Hodgkin lymphoma cell lines. Blood. 2008;112:1424–1433. doi: 10.1182/blood-2008-01-133769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Catley L, Weisberg E, Kiziltepe T, Tai YT, Hideshima T, Neri P, Tassone P, Atadja P, Chauhan D, Munshi NC, Anderson KC. Aggresome induction by proteasome inhibitor bort-ezomib and alpha-tubulin hyperacetylation by tubulin deacetylase (TDAC) inhibitor LBH589 are synergistic in myeloma cells. Blood. 2006;108:3441–3449. doi: 10.1182/blood-2006-04-016055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Corcoran LJ, Mitchison TJ, Liu Q. A novel action of histone deacetylase inhibitors in a protein aggresome disease model. Current Biology. 2004;14:488–492. doi: 10.1016/j.cub.2004.03.003. [DOI] [PubMed] [Google Scholar]
  5. Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunological Reviews. 2009;229:173–191. doi: 10.1111/j.1600-065X.2009.00766.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Drexler HG. Recent results on the biology of Hodgkin and Reed-Sternberg cells. II. Continuous cell lines. Leukaemia & Lymphoma. 1993;9:1–25. doi: 10.3109/10428199309148499. [DOI] [PubMed] [Google Scholar]
  7. Fournel M, Bonfils C, Hou Y, Yan PT, Trachy-Bourget MC, Kalita A, Liu J, Lu AH, Zhou NZ, Robert MF, Gillespie J, Wang JJ, Ste-Croix H, Rahil J, Lefebvre S, Moradei O, Delorme D, Macleod AR, Besterman JM, Li Z. MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo. Molecular Cancer Therapeutics. 2008;7:759–768. doi: 10.1158/1535-7163.MCT-07-2026. [DOI] [PubMed] [Google Scholar]
  8. Georgakis GV, Li Y, Rassidakis GZ, Martinez-Valdez H, Medeiros LJ, Younes A. Inhibition of heat shock protein 90 function by 17-allylamino-17-demethoxy-geldanamycin in Hodgkin's lymphoma cells down-regulates Akt kinase, dephosphorylates extracellular signal-regulated kinase, and induces cell cycle arrest and cell death. Clinical Cancer Research. 2006;12:584–590. doi: 10.1158/1078-0432.CCR-05-1194. [DOI] [PubMed] [Google Scholar]
  9. Gloghini A, Buglio D, Khaskhely NM, Georgakis G, Orlowski RZ, Neelapu SS, Carbone A, Younes A. Expression of histone deacetylases in lymphoma: implication for the development of selective inhibitors. British Journal of Haematology. 2009;147:515–525. doi: 10.1111/j.1365-2141.2009.07887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guo JJ, Li QL, Zhang J, Huang AL. Histone deacetylation is involved in activation of CXCL10 upon IFNgamma stimulation. Molecules and Cells. 2006;22:163–167. [PubMed] [Google Scholar]
  11. Hauser AT, Jung M. Assays for histone deacetylases. Current Topics in Medicinal Chemistry. 2009;9:227–234. doi: 10.2174/156802609788085269. [DOI] [PubMed] [Google Scholar]
  12. Kim HS, Lee MS. STAT1 as a key modulator of cell death. Cellular Signalling. 2007;19:454–465. doi: 10.1016/j.cellsig.2006.09.003. [DOI] [PubMed] [Google Scholar]
  13. Lane AA, Chabner BA. Histone deacetylase inhibitors in cancer therapy. Journal of Clinical Oncology. 2009;27:5459–5468. doi: 10.1200/JCO.2009.22.1291. [DOI] [PubMed] [Google Scholar]
  14. Marks PA, Xu WS. Histone deacetylase inhibitors: potential in cancer therapy. Journal of Cellular Biochemistry. 2009;107:600–608. doi: 10.1002/jcb.22185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mottok A, Renne C, Willenbrock K, Hansmann ML, Brauninger A. Somatic hypermutation of SOCS1 in lymphocytepredominant Hodgkin lymphoma is accompanied by high JAK2 expression and activation of STAT6. Blood. 2007;110:3387–3390. doi: 10.1182/blood-2007-03-082511. [DOI] [PubMed] [Google Scholar]
  16. Peh SC, Kim LH, Poppema S. TARC, a CC chemokine, is frequently expressed in classic Hodgkin's lymphoma but not in NLP Hodgkin's lymphoma, T-cell-rich B-cell lymphoma, and most cases of anaplastic large cell lymphoma. American Journal of Surgical Pathology. 2001;25:925–929. doi: 10.1097/00000478-200107000-00011. [DOI] [PubMed] [Google Scholar]
  17. Redmond WL, Weinberg AD. Targeting OX40 and OX40L for the treatment of autoimmunity and cancer. Critical Reviews in Immunology. 2007;27:415–436. doi: 10.1615/critrevimmunol.v27.i5.20. [DOI] [PubMed] [Google Scholar]
  18. Vire B, de Walque S, Restouin A, Olive D, Van Lint C, Collette Y. Anti-leukemia activity of MS-275 histone deacetylase inhibitor implicates 4-1BBL/4-1BB immunomodulatory functions. PLoS ONE. 2009;4:e7085. doi: 10.1371/journal.pone.0007085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wang C, Lin GH, McPherson AJ, Watts TH. Immune regulation by 4-1BB and 4-1BBL: complexities and challenges. Immunological Reviews. 2009;229:192–215. doi: 10.1111/j.1600-065X.2009.00765.x. [DOI] [PubMed] [Google Scholar]
  20. Weihrauch MR, Manzke O, Beyer M, Haverkamp H, Diehl V, Bohlen H, Wolf J, Schultze JL. Elevated serum levels of CC thymus and activation-related chemokine (TARC) in primary Hodgkin's disease: potential for a prognostic factor. Cancer Research. 2005;65:5516–5519. doi: 10.1158/0008-5472.CAN-05-0100. [DOI] [PubMed] [Google Scholar]
  21. Weniger MA, Melzner I, Menz CK, Wegener S, Bucur AJ, Dorsch K, Mattfeldt T, Barth TF, Moller P. Mutations of the tumour suppressor gene SOCS-1 in classical Hodgkin lymphoma are frequent and associated with nuclear phospho-STAT5 accumulation. Oncogene. 2006;25:2679–2684. doi: 10.1038/sj.onc.1209151. [DOI] [PubMed] [Google Scholar]
  22. Xu WS, Parmigiani RB, Marks PA. Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene. 2007;26:5541–5552. doi: 10.1038/sj.onc.1210620. [DOI] [PubMed] [Google Scholar]
  23. Younes A, Pro B, Fanale M, McLaughlin P, Neelapu S, Fayad L, Wedgwood A, Buglio D, Patterson T, Dubay M, Li Z, Martell RE, Ward MR, Bociek RG. Isotype-Selective HDAC Inhibitor MGCD0103 Decreases Serum TARC Concentrations and Produces Clinical Responses in Heavily Pretreated Patients with Relapsed Classical Hodgkin Lymphoma (HL) Blood (ASH Annual Meeting Abstracts) 2007;110:2566. [Google Scholar]
  24. Yuan Z, Rezai-Zadeh N, Zhang X, Seto E. Histone deacetylase activity assay. Methods in Molecular Biology. 2009;523:279–293. doi: 10.1007/978-1-59745-190-1_19. [DOI] [PubMed] [Google Scholar]
  25. Zheng B, Fiumara P, Li YV, Georgakis G, Snell V, Younes M, Vauthey JN, Carbone A, Younes A. MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival. Blood. 2003;102:1019–1027. doi: 10.1182/blood-2002-11-3507. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

2

Fig S1. Effect of the class-I selective HDAC inhibitor MGCD0103 and two pan HDAC inhibitors (vorinostat and trichostatin-A on purified HDAC 1-10 enzymatic activity in cell-free assays.

Fig S2. Biogroups defined in NextBio are collections of genes sharing a specific biological function or pathway and could consist of signaling and metabolic pathways, protein families, gene ontologies, as well as other relevant gene sets.

Fig S3. GEP focusing on the TNFRSF pathway in the L428 cells.

Fig S4. (A) The effect of MGCD0103 on TNFRSF8 expression is confirmed by RT-PCR. (B) Summary results demonstrating the effect of MGCD0103 (1 μmol/l) on TNFRSF8 surface expression as determined by FACS analysis.

Fig S5. An example of the effect of MGCD0103 on inflammatory cytokine and chemokines using gene expression profiling (GEP) in the KM-H2 cell line.

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