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Cancer Therapy: Preclinical

JNJ-26481585, a Novel “Second-Generation” Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

Janine Arts, Peter King, Ann Mariën, Wim Floren, Ann Beliën, Lut Janssen, Isabelle Pilatte, Bruno Roux, Laurence Decrane, Ron Gilissen, Ian Hickson, Veronique Vreys, Eugene Cox, Kees Bol, Willem Talloen, Ilse Goris, Luc Andries, Marc Du Jardin, Michel Janicot, Martin Page, Kristof van Emelen and Patrick Angibaud
DOI: 10.1158/1078-0432.CCR-09-0547 Published November 2009

Abstract

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a “second-generation” class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy.

Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors.

Results: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L).

Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising “second-generation” HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):6841–51)

Keywords
  • HDAC
  • JNJ-26481585
  • inhibitor
  • antitumor agent
  • second-generation

Translational Relevance

Histone deacetylase (HDAC) inhibitors have shown clinical activity in hematologic malignancies, but their activity in solid tumor indications has been somewhat limited. We hypothesized that this could be related to the transient pharmacodynamic effects of “first-generation” HDAC inhibitors in tumor cells. This hypothesis is further supported by the observation that HDAC inhibitors need to be given chronically and prolonged exposure to HDAC inhibitors seems essential to achieve effective tumor cell death. Here, we identified JNJ-26481585, a “second-generation” HDAC inhibitor with prolonged pharmacodynamic response in vivo. In agreement with the hypothesis, JNJ-26481585 showed superior efficacy compared with both standard of care agents and first-generation HDAC inhibitors in preclinical tumor models. These studies suggest that an HDAC inhibitor with continuous pharmacodynamic activity may show activity in solid tumor malignancies. JNJ-26481585 is currently being evaluated in the clinic in tumors known to be driven by high class I HDAC activity.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, terminal differentiation, and apoptosis in a broad spectrum of human tumor cell lines in vitro, and have antiangiogenic and antitumor activity in human xenograft models (1–5). Several HDAC inhibitors are in clinical development where activity has been observed mainly in hematologic malignancies. This has led to the recent approval of vorinostat (suberoylanilide hydroxamic acid) for the treatment of cutaneous T-cell lymphoma (6).

Four classes of HDACs have been identified: classes I, II, III, and IV. Most HDAC inhibitors under clinical development act on three distinct classes of HDAC enzymes: class I, comprising HDAC1-3 and HDAC8; class IIa, comprising HDAC4, HDAC5, HDAC7, HDAC9; and class IIb, comprising HDAC6 and HDAC10. HDAC11 is the sole member of class IV HDACs. Inhibition of class I HDACs results in the acetylation of nuclear histone proteins, which affects tertiary chromatin structure and leads to altered expression of genes involved in cell proliferation, apoptosis, and differentiation. Class I HDAC activity is key for uncontrolled proliferation of cancer cells, because downregulation of HDAC1 and HDAC3 expression results in increased histone acetylation and inhibition of tumor cell proliferation (7). In contrast to the class I HDAC family members, class II HDACs are not as widely involved in processes that control survival of solid cancer cells. Downregulation of class IIa members HDAC4 and HDAC7 in HeLa cells using siRNA technology did not result in decreased proliferation (7), and inhibition of the class IIb enzyme HDAC6 similarly did not widely affect tumor cell survival. HDAC6, however, is a deacetylase for tubulin and Hsp90. HDAC6 inhibitors decrease cell motility (8, 9) and deplete oncogenic Hsp90 client proteins, resulting in potentiation of therapeutics such as paclitaxel, trastuzumab, and bortezomib (10, 11).

We previously identified R306465, a highly potent class I selective HDAC inhibitor, showing oral antitumor activity in human tumor–bearing mice (12). Although R306465 showed antitumoral activity in nonestablished tumor models in vivo, its activity was found to be modest when treating immunodeficient mice carrying pre-established tumors. Pharmacodynamic studies revealed that R306465 has a short half-life and only transiently induces histone acetylation in vivo, a feature shared with a number of other HDAC inhibitors in clinical development. Vorinostat, for example, has an apparent plasma half-life of 91 to 127 minutes and the induction of H3 acetylation in circulating peripheral blood mononuclear cells was found to be transient (13, 14).

To identify a “second-generation” HDAC inhibitor with improved single agent antitumoral efficacy, we aimed to design a molecule with high potency toward class I HDACs, showing a prolonged pharmacodynamic response in vivo. We, therefore, developed an experimental tumor model which allows noninvasive real-time analysis of HDAC1 activity in tumor tissue (15). In this model, human A2780 ovarian carcinoma cells have been engineered with a reporter gene construct encoding the fluorescent ZsGreen protein, whose expression is under the control of the p21waf1,cip1 promoter, a gene constitutively repressed by the HDAC1 protein (16). In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585, which after once daily oral administration induced continuous histone H3 acetylation and complete tumor growth inhibition in human HCT116 colon tumors.

The potent antitumoral activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of human solid and hematologic malignancies.

Materials and Methods

Compounds

JNJ-26481585, R306465 and vorinostat (Fig. 1D), panobinostat, CRA-024781, and mocetinostat were synthesized according to published methods. All compounds were dissolved in DMSO as 5 mmol/L stock solutions and kept at room temperature.

Fig. 1.
Fig. 1.

Identification of JNJ-26481585 as a potent HDAC1 inhibitor in p21waf1,cip1 ZsGreen tumors in vivo. Human A2780-p21waf1,cip1ZsGreen ovarian tumors cells were injected s.c. into the inguinal region of male athymic nu/nu CD-1 mice. When palpable tumors were obtained, mice were treated p.o. once daily for 3 d, with JNJ-26481585 (10 mg/kg i.p. or 40 mg/kg p.o.), R306465 (40 mg/kg p.o.), vorinostat (200 mg/kg p.o.), or vehicle (20% hydroxypropyl-β-cyclodextrin). A, fluorescent images of A2780-p21waf,cip1ZsGreen tumors from four representative mice collected 24 h following the last treatment. B, for optimal visualization of the ZsGreen-fluorescence, the channels of the initial triple channel images are separated in two rows combining ZsGreen (green) and blood vessels (CD31; red) in the top row and CD31 (red) and Hoechst in the bottom row. The latter combination shows that the images were taken from areas that have a similar density of tumor cells. C, localization of fluorescence in xenograft tissue. The actin staining (red) marks the boundaries of all tumor cells. D, chemical structure of JNJ-26481585, R306465, and vorinostat.

HDAC activity assays

Recombinant HDAC activity assays were done by Reaction Biology Corporation. In all cases, full-length HDAC proteins were expressed using baculovirus-infected Sf9 cells. In addition, HDAC3 was coexpressed as a complex with human NCOR2. For assessing activity of HDAC1-containing cellular complexes, immunoprecipitated HDAC1 complexes were incubated with an [3H]acetyl- labeled fragment of histone H4 peptide [biotin-(6-aminohexanoic)Gly-Ala-(acetyl[3H])Lys-Arg-His-Arg-Lys-Val-NH2; Amersham Pharmacia Biotech] as described (12). Equal amounts of HDAC1 were immunoprecipitated as indicated by Western blot analysis. HDAC1 activity results are presented as mean ± SD of three independent experiments on a single lysate.

Cell proliferation and apoptosis assays

All cell lines were obtained from American Type Culture Collection and cultured according to instructions. The effect of HDAC inhibitors on cell proliferation was measured using an MTT as described (12). Proliferation of non–small cell lung carcinoma (NSCLC) cell lines was assessed using an Alamar Blue–based assay as described (12). For proliferation of hematologic cell lines, cells were incubated for 72 h and the cytotoxic activity was evaluated by MTS assay. Data are presented as mean IC50 or IC40 ± SD of at least three independent experiments. For apoptosis assays, human tumor cells were incubated for 24, 48, and 96 h with JNJ-26481585 at the indicated concentrations. Cells were stained for Annexin V and 7-AAD, according to the manufacturer's instructions and analyzed (Guava PCA-96 Nexin kit, Guava Technologies). The number of apoptotic and necrotic cells was expressed as a percentage of the total number of cells present in the well. Total cell number was expressed as a percentage of control, and the percentage of apoptotic/necrotic cells present in the absence of compound was subtracted from all values. All results shown are an average of three independent experiments (± SD).

Western blot analysis

Human A2780 ovarian carcinoma cells were incubated with the indicated concentrations of JNJ-26481585, CRA-024781, and mocetinostat. Total cell lysates were prepared using hot lysis buffer containing 1% SDS in 10 mmol/L Tris (pH 7.4), supplemented with 1 mmol/L sodium orthovanadate. Cleared cell lysates were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Levels of acetylated H3 (AcH3) and H4 histones (Upstate/Millipore), histone H4-acetyl K16 (Abcam), histone H4-trimethyl K20 (Abcam), total H3 protein (Abcam), E-Cadherin (Abcam), p21waf1,cip1 (BD Transduction Laboratories), total tubulin (Sigma), acetylated tubulin (Sigma), Hsp70 (Stressgen), and c-Raf (BD Transduction Laboratories) protein were detected using the appropriate antibodies. To control for equal loading, blots were stripped and reprobed with anti-actin (Oncogene Research Products) or anti-lamin B1 (Zymed, Invitrogen) antibodies. As secondary antibodies, horseradish peroxidase–labeled anti-mouse (Santa Cruz Biotechnology), horseradish peroxidase–labeled anti-rabbit (Zymed), fluorochrome-labeled anti-mouse (Rockland), or fluorochrome-labeled anti-rabbit (Invitrogen Molecular Probes) were used. Protein-antibody complexes were then visualized by chemiluminescence (Pierce Chemical Co.) or fluorescence (Odyssey, Li-Cor Biosciences) according to manufacturer's instructions.

In vivo xenograft studies

JNJ-26481585, R306465, and vorinostat were formulated at 2 mg/mL in 20% hydroxypropyl-β-cyclodextrin (final pH 8.7). 5-Fluorouracil (5-FU) was formulated in 20% hydroxypropyl-β-cyclodextrin (pH 4.4). All mice used in the in vivo studies were treated according to the ethical guidelines prescribed by United Kingdom Coordinating Committee on Cancer Research. HCT116 human colon carcinoma cells were injected s.c. (107 cells/200 μL) into the inguinal region of athymic male NMRI nu/nu mice purchased from Janvier. Tumor size was determined using caliper measurement and tumor volume was determined by using the formula: TV = (a2 × b)/2 (in which a represents the width and b the length). The human colon carcinoma cell line C170HM2 was derived originally from a primary tumor. C170HM2 cell suspensions (1.5 × 106 cells) were injected into the peritoneal cavity of male MFI nude mice (bred within the Cancer Studies Unit at the University of Nottingham, United Kingdom) in 1 mL of 0.9% sodium chloride (pH 7.3). The liver was exposed, excised, and analyzed as described (17). Statistical analysis was done using Wilcoxon Mann-Whitney analysis with P < 0.05 considered statistically significant. To generate the xenograft model allowing imaging of HDAC1 activity, an HDAC1-responsive p21waf1,cip1 promoter construct, the −1300 to +88 region of the p21waf1,cip1 promoter, was cloned into pGL3-basic-ZsGreen and stably transfected into A2780 ovarian carcinoma cells (15). For in vivo analysis, mice carrying A2780-p21waf1,cip1ZsGreen tumors were treated from day 12 after grafting both orally (p.o.) and i.p. once daily for 4 d with solvent or the indicated dose of HDAC inhibitors. Tumors were evaluated for fluorescence by an automated Whole-Body Imaging system (12, 15).

Immunohistochemistry

HCT116 colon xenograft tissue and mouse skin were embedded in paraffin and further processed for immunofluorescence staining. The following primary antibodies were used: acetylated histone H3 (Upstate/Millipore), histone H4-acetyl K16 (Abcam), or histone H4-trimethyl K20 (Abcam). After overnight incubation with the primary antibody, an appropriate goat-specific CY3-conjugated secondary antibody (Jackson Immunoresearch Laboratories) was used. For negative controls, the primary antibody was omitted. For vessel localization, tumors were collected using transcardial perfusion fixation with 4% paraformaldehyde. After cryoprotection, cryosections of 10-μm thickness were made and mounted on glass slides. The xenografts were stained for actin using BODIPY 558/568-phalloidin (Invitrogen Molecular Probes) and the vessels were stained using CD-31 antibody (BD Transduction Laboratories) and a goat anti-rat secondary antibody coupled to Cy3 (Jackson Immunoreasearch Laboratories). Images were taken using a Zeiss Axioplan 2 microscope equipped with an Axiocam HRm camera and Axiovision imaging software (Zeiss).

In vivo pharmacokinetic and pharmacodynamic analysis

Tumor material from mouse xenografts was isolated and homogenized using sonication. H3 acetylation was determined by ELISA or MSD (Meso Scale Discovery), according to the manufacturer's instructions. Briefly, the anti-histone PAN antibody (Chemicon/Millipore) was spotted onto the base of a 96-well MSD Standard Bind plate. After blocking, the xenograft sample [2.5 μg/25 μL 10 mmol/L Tris (pH 7.4) supplemented with 1 mmol/L sodium orthovanadate] or calibrator sample (Upstate/Millipore) was added and the array was incubated with anti-histone H3 antibody (Upstate/Millipore). Assay results were read using the MSD Sector Imager 6000.

The model-based analysis of the pharmacokinetic and pharmacodynamic data were done using S-PLUS version 7.0 (Insightful Corporation) and NONMEM version 5 (ICON). The time course of drug concentration in the plasma over the duration of the study was described on the basis of a two-compartment disposition model. This concentration versus time profile can be described by the following equation: [Ct = I* (A · e · + B · e−β·t) eq. PKPD1]. Ct is the concentration of JNJ-26481585 in the plasma at time t since the first dose, I is the administration schedule of the compound (10 mg/kg every 24 h for 21 d), * denotes convolution, whereas A and B, and α and β, are the coefficients and the exponents describing the disposition function of JNJ-26481585, respectively. The time course of AcH3 response in the tumor at day 1 and day 7 was described on the basis of an indirect response pharmacodynamic model (18). In the model, the level of H3 acetylation at baseline is the net effect of histone acetylation and deacetylation. Treatment with JNJ-26481585 disrupts this balance by inhibition of deacetylation. The net effect is an increased level of H3 acetylation. The process is described by the following equation:dAcH3dt=KHAC+KHDAC·AcH3,t·(1−CJNJ,tIC50+CJNJ,t)KHAC is the presumed zero-order rate constant of histone acetylation and KHDAC is the first-order rate constant of histone deacetylation in the absence of JNJ-26481585. AcH3,t is the concentration of acetylated histone on the tumor tissue at time t, CJNJ,t is the plasma concentration of JNJ-26481585 at time t, and IC50 is the plasma concentration of JNJ-26481585 resulting in 50% inhibition of rate of histone deacetylation.

Results

Identification of JNJ-26481585 as a potent HDAC1 inhibitor in vivo

To identify an HDAC inhibitor with improved potency and prolonged pharmacodynamic response in tumor tissue, we preselected 140 pyrimidyl-hydroxamic acid analogues that were all potent HDAC inhibitors in HeLa nuclear extracts in vitro (IC50, 0.05-100 nmol/L). We subsequently analyzed all these promising compounds in vivo in an HDAC1-responsive A2780 ovarian tumor screening model. In this model, the HDAC1-regulated p21waf1,cip1 promoter controls a fluorescent ZsGreen protein. We previously showed that induction of tumor fluorescence fully predicts tumor growth inhibition after dosing experimental agents for 3 days only (15). As shown in Fig. 1A, JNJ-26481585 was the only compound identified that induced a bright and intense fluorescence in the tumor xenografts after dosing for 3 days at its maximal tolerated dose (10 mg/kg i.p. and 40 mg/kg p.o.). The bright fluorescence was in contrast to the more weak induction observed with other clinical HDAC inhibitors such as R306465 (40 mg/kg, oral; ref. 12) and vorinostat (200 mg/kg, p.o.), which were also administered at their maximal tolerated dose. The maximal tolerated doses of JNJ-26481585 and vorinostat were determined, after a 20-day dosing period, as the doses that resulted in <15% weight loss and no lethality (data not shown). In JNJ-26481585–treated tumors, fluorescence was not uniformly distributed throughout the tissue (Fig. 1B and C). Generally, focal spots of high fluorescence were surrounded by areas with weaker intensity. The areas of these high intensity spots contained from a few to hundreds of cells and did not colocalize with tumor vasculature (Fig. 1B). There was no difference in fluorescence between the peripheral and central parts of the tumor, indicating exposure of the drug and/or a biological response throughout the entire tumor (data not shown).

To investigate the reason for the exquisite potency of JNJ-26481585 in activating the HDAC1-regulated p21waf1, cip1 promoter in vivo, we investigated inhibition of HDAC1 enzyme activity by immunoprecipitating HDAC1 complexes from A2780 ovarian carcinoma cells in vitro. JNJ-26481585 inhibited HDAC1 complexes with an IC50 value of 0.16 ± 0.02 nmol/L (average ± SD, n = 3), which is 20-fold more potent than R306465 (IC50, 3.31 ± 0.78 nmol/L) and 530-fold more potent than vorinostat (IC50, 85 ± 16 nmol/L). HDAC1 suppresses p21waf1,cip1 gene expression by deacetylating nuclear histone proteins, which affects tertiary chromatin structure (16). We, therefore, measured the kinetics of histone H3 acetylation in the A2780 xenograft tissue in vivo, to assess the impact of the high HDAC1 potency on the duration of the pharmacodynamic response. Immunodeficient mice carrying A2780 ovarian tumors were treated once daily with JNJ-26481585 (10 mg/kg, i.p.) and H3 acetylation in the xenograft was analyzed using a quantitative ELISA. As shown in Fig. 2, JNJ-26481585 induces potent tumor H3 acetylation (both after the first dose and at day 7) and the time course of this drug effect is delayed when compared with the time course of JNJ-26481585 exposure in the plasma: the maximum AcH3 response is observed 2 to 5 hours postdose, whereas the maximum exposure in plasma is observed at 30 minutes postdose. Importantly, after dosing JNJ-26481585 for 7 days, there is a higher basal level of H3 acetylation in the tumor than on day 1. This indicates that the AcH3 response further accumulates between day 1 and day 7, and H3 acetylation in the tumor tissue is continuously induced, although drug plasma exposure does not show accumulation between day 1 and day 7. Tumor concentrations of JNJ-26481585 were measured on days 14 and 21 and show that there is continuous exposure of HDAC1 to JNJ-26481585 in tumor tissue (Supplementary Fig. S7). The short plasma half-life of JNJ-26481585 in nude mice is in agreement with the observed extensive rodent-specific first-pass metabolism. Therefore, subsequent antitumor studies with JNJ-26481585 in nude mice were done i.p., the optimal route of administration for this compound in rodents.

Fig. 2.
Fig. 2.

JNJ-26481585 induces continuous H3 acetylation in tumor tissue in vivo. Human A2780 ovarian tumor cells (107 cells/mouse) were injected s.c. into the inguinal region of male athymic nu/nu CD-1 mice. When palpable tumors were obtained, mice were treated once daily with vehicle (10% hydroxypropyl-β-cyclodextrin) or JNJ-26481585 at 10 mg/kg i.p., and tumor and plasma was harvested at day 1 and at day 7 at the indicated time points (5 mice/point). Levels of AcH3 were determined using a quantitative ELISA (300 ng tumor protein/well) and described on the basis of an indirect response pharmacodynamic model as described in the Materials and Methods section. Time course for measurement of drug concentration in mouse plasma (○) and AcH3 in tumor tissue (A2780 ovarian cell line, •) are shown (samples taken at 0, 30 min, 1, 2, 4, 8, and 24 h postdose).

The time course of AcH3 response in the tumor at day 1 and day 7 could be described on the basis of an indirect response pharmacodynamic model (18). In this model, the drug effect (inhibition of the deacetylation of histone) is integrated with the rates of both histone acetylation and deacetylation (see Materials and Methods section). Figure 2 shows that AcH3 response continues to increase with dosing of JNJ-26481585; then is expected to return to pretreatment values ∼21 days after treatment termination. KHDAC, the first-order rate constant of histone deacetylation in the absence of JNJ-26481585, was found to be highly different between different tumors, being 19 U/h in A2780 ovarian xenografts, but much lower (1.8 U/h) in HCT116 colon xenografts (data not shown). The plasma concentration of JNJ-26481585 resulting in 50% inhibition of rate of histone deacetylation (IC50), however, was comparable across tumors, being 5.9 and 7.9 ng/mL in A2780 ovarian and HCT116 colon xenografts, respectively (14 and 18 nmol/L). These data indicate that JNJ-26481585 shows comparable potency across tumor types, independent of their basal level of HDAC activity.

The observation that JNJ-26481585 causes prolonged H3 acetylation in the tumor tissue, despite its short plasma half-life, is in contrast to the effects of less potent HDAC1 inhibitors. For example, R306465 was previously found to cause only a short transient induction of H3 acetylation in tumor tissue at 1 and 4 hours postdose, returning to baseline levels at 8 hours (data not shown).

HDAC isotype specificity of JNJ-26481585

To characterize the inhibition of other HDAC family members by JNJ-26481585, we tested its potency toward a broad panel of recombinant HDAC enzymes in vitro. As shown in Table 1, JNJ-26481585 shows activity toward all HDAC enzymes tested with highest potency in vitro observed toward recombinant HDAC1 (IC50, 0.11 ± 0.03 nmol/L), which is comparable with the potency observed toward HDAC1-immunoprecipitated complexes from tumor cells (IC50, 0.16 ± 0.02 nmol/L). Lowest in vitro potency was observed toward HDAC6, 7 and 9 (IC50, 32.1-119 nmol/L). As shown in Fig. 3A and B, unsupervised clustering of isotype specificity with a panel of other HDAC inhibitors in clinical development revealed that in vitro JNJ-26481585 and its predecessor R306465 show a class I–specific inhibition profile that most resembles that of the fully class – specific benzamide mocetinostat (r = 0.75; ref. 19), with the distinction that JNJ-26481585 is 100-fold more potent than mocetinostat. JNJ-26481585 is most distinct from vorinostat (r = 0.26) and CRA-024781 (r = 0.40; ref. 20).

View this table:
Table 1.

Activity toward tested HDAC enzymes (nM ± SD)

Fig. 3.
Fig. 3.

JNJ-26481585 inhibits HDAC isozymes in vitro. Recombinant HDAC activity assays were done by Reaction Biology Corporation. In all cases, full-length HDAC proteins were expressed in baculovirus-infected sf9 cells. In addition, HDAC3 was coexpressed as a complex with human NCOR2. Enzymes were incubated with the indicated compounds at concentrations ranging from 0.1 nmol/L to 10 μmol/L. A, graphs showing Pearson correlation. B, two-way hierarchical clustering of IC50s, where both compounds and HDAC enzymes were clustered based on their IC50 values. The clustering was generated by Ward's clustering with Pearson correlation using the package plots implemented in R version 2.7.1.

Analyzing specificity profiles based on inhibition of isolated recombinant enzymes can be misleading, especially for HDACs, which function as large multiprotein complexes. We, therefore, studied inhibition by analyzing HDAC substrate acetylation in A2780 ovarian carcinoma cells. JNJ-26481585 induced H3 and H4 acetylation at concentrations as low as 30 to 100 nmol/L (Fig. 4), indicating high potency toward class I HDACs in tumor cells. JNJ-26481585 also induced acetylation and trimethylation at Lys16 and Lys20 of histone H4, respectively (Fig. 4). Loss of these modifications is a common hallmark of human cancer (21). Genes specifically silenced by HDAC1, such as p21waf1,cip1 and E-cadherin, were induced with similar high potency, confirming potent HDAC1 inhibition in tumor cells. Surprisingly, despite modest in vitro potency against recombinant HDAC6, in A2780 tumor cells, JNJ-26481585 also showed high cellular potency toward HDAC6. JNJ-26481585 induced acetylation of HDAC6 substrate tubulin at concentrations as low as 30 to 100 nmol/L. Similarly, Hsp90 activity is inhibited, as evidenced by induction of Hsp70 and loss of Hsp90 client c-Raf. Therefore, in tumor cells, JNJ-26481585 shows equipotent inhibition of HDAC1 and HDAC6 and behaves like a true pan-HDAC inhibitor.

Fig. 4.
Fig. 4.

JNJ-26481585 is a potent pan-HDAC inhibitor in tumor cells. Human A2780 ovarian carcinoma cells were incubated with the indicated concentrations of JNJ-26481585, R306465, vorinostat, panobinostat, mocetinostat, and CRA-024781 for 24 h. Total cell lysates were prepared and analyzed by SDS-PAGE. Levels of acetylated histone H3 (H3 Ac), H4 (H4 Ac), and tubulin; levels of histone H4-acetyl K16 (H4-K16 Ac) and histone H4-trimethyl K20 (H4-K20 Me3); total levels of histone H3, tubulin, p21waf1, cip1, Hsp70, E-cadherin, and c-Raf protein were detected by enhanced chemiluminescence detection. To control for equal loading, blots were stripped and reprobed with antibodies against actin or nuclear protein Lamin B1 to control for the efficiency of extraction of nuclear proteins. Shown is a representative of three experiments in comparison with previously published results for R306465, vorinostat, and panobinostat (12).

We further compared the specificity profile of JNJ-26481585 with other HDAC inhibitors in clinical development (Fig. 4). Vorinostat has been shown to induce histone H3 acetylation only at 1 to 3 μmol/L, whereas tubulin acetylation and Hsp70 induction were already evident at 100 nmol/L, indicating a 10-fold higher potency for HDAC6 for this agent (12). This is in line with the higher relative potency for HDAC6 with vorinostat compared with JNJ-26481585 observed on these enzymes in vitro. Also, class I–specific benzamide mocetinostat induced histone H3 acetylation at 3 μmol/L, but did not affect HDAC6 substrates. Finally, in agreement with previous publications, the hydroxamic acid–based HDAC inhibitors CRA-024781 and panobinostat (12) both also inhibited HDAC1/HDAC6 at similar concentrations, because both substrates were acetylated at 100 and 300 nmol/L (10).

JNJ-26481585 has broad spectrum antiproliferative activity against solid and hematologic cancer cell lines and induces apoptosis

Class I HDACs such as HDAC1 have been shown to be key for tumor cell proliferation (7). As JNJ-26481585 shows such high potency in tumor cells versus class I HDACs, we, therefore, investigated the antiproliferative effects of JNJ-26481585 in a broad panel of human tumor cell lines from both solid and hematologic origin. As indicated in Fig. 5A, JNJ-26481585 inhibited cell proliferation in all lung, breast, colon, prostate, brain, and ovarian tumor cell lines tested, with IC50 values ranging from 3.1 to 246 nmol/L. We also observed that JNJ-26481585 exhibited a more potent antiproliferative effect than vorinostat, R306465, panobinostat, CRA-24781, or mocetinostat in various human cancer cell lines tested (Supplementary Fig. S8). This is in agreement with its high relative potency toward class I HDACs (HDAC1/2) compared with other HDAC inhibitors (Fig. 3).

Fig. 5.
Fig. 5.

JNJ-26481585 induces tumor cell apoptosis. Human tumor cell lines were seeded and after 24 h cells incubated with JNJ-26481585 at 3, 10, 30, 100, and 300 nmol/L. A, the number of viable cells after a 4-d incubation period was assessed using a standard MTT colorimetric assay or Alamar Blue assay. Values represent mean ± SD of three independent experiments. B, for hematologic tumor cell lines, IC40 values (concentrations leading to 40% inhibition of cell proliferation) were determined. ALL, acute lymphoblastic leukemia; AML, Acute Myeloid Leukemia; CLL, chronic lymphoblastic leukemia; and CML, chronic myeloid leukemia. C, the indicated cell lines were stained with Annexin V and propidium iodide after 48 h of incubation with JNJ-26481585 and analyzed by fluorescence-activated cell sorting.

This antiproliferative effect was not dependent on p53 genotypic status, or Ras mutational status in colon and lung tumor cells (data not shown). Similarly, as shown in Fig. 5B, JNJ-26481585 inhibited the proliferation with comparable potency of acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, and myeloma tumor cells (IC40, 4.5-166 nmol/L). To investigate whether the antiproliferative effect of JNJ-26481585 in A2780 ovarian tumor cells was due to induction of cell cycle arrest or cell death, fluorescence-activated cell sorting analysis was done. As shown in Fig. 5C, after 48 hours of incubation, in all cell lines investigated, JNJ-26481585 treatment at 3 to 300 nmol/L caused a significant increase in the percentage of cells positive for Annexin V in a concentration-dependent manner indicative of apoptosis. An increase in the number of necrotic cells was also observed, which likely represents apoptotic cells in later stages of cell death.

Antitumor efficacy of JNJ-26481585

As JNJ-26481585 potently induces tumor cell apoptosis, and shows a continuous pharmacodynamic response in tumor tissue in vivo, we subsequently investigated whether these properties indeed translated into improved efficacy in preclinical tumor in vivo models. As shown in Fig. 6A, JNJ-26481585 administered continuously for 14 days (once daily, 10 mg/kg i.p.) strongly inhibited the growth of large pre-established HCT116 colon xenografts (320 ± 10 mm3 at start of treatment). At the end of the study, JNJ-26481585 inhibited tumor volume by 76% (treated versus control = 24), which is superior to the activity of the clinical standard of care agent 5-FU (41% inhibition). The first-generation HDAC inhibitor vorinostat showed only modest activity in the model (26% inhibition). In agreement with its low antitumor potency, vorinostat only slightly increased H3 acetylation levels at 4 hours postdose (0.03 ± 0.02 ng/μg protein), whereas JNJ-26481585 showed a more potent effect (0.22 ± 0.07 ng/μg protein; data not shown). A subsequent dose-response study to further explore the potency of JNJ-26481585 showed similar tumor growth inhibition at 5 mg/kg compared with 20 mg/kg (87% and 93% inhibition, respectively, Fig. 6B), whereas half-maximal inhibition was obtained at the low dose of 2.5 mg/kg in the pre-established setting (69% inhibition).

Fig. 6.

JNJ-26481585 potently inhibits growth of colorectal cancer xenografts and colon metastases in the liver. A and B, HCT116 colon carcinoma cells were injected s.c. into the inguinal region of NMRI nude mice (106 cells/mouse). Subcutaneous tumors were measured at indicated time points (twice a week) throughout the study, and results represented as the median relative tumor volume (± variation; mm3) of 10 animals per group. A, mice were dosed starting at day 11 and treated at the indicated time points with JNJ-26481585 (10 mg/kg once daily, i.p.;

Graphic
), vorinostat (100 mg/kg twice daily p.o.;
Graphic
), 5-FU (100 mg/kg once weekly i.p.;
Graphic
), or vehicle (20% hydroxypropyl-β-cyclodextrin, □). B, mice were dosed starting at day 6 (tumor volume, 161 ± 5 mm3) and treated once daily i.p. with vehicle (hydroxypropyl-β-cyclodextrin; □) or vehicle containing JNJ-26481585 at either 2.5 mg/kg (
Graphic
), 5 mg/kg (
Graphic
), 10 mg/kg (
Graphic
), or 20 mg/kg (
Graphic
) for 21 d. C, H3 acetylation (H3 Ac), histone H4-trimethyl K20 (H4-K20 Me3), and histone H4-acetyl K16 (H4-K16 Ac) immunofluorescent staining of HCT116 colon xenograft tissue, epidermis, and hair follicle cells with and without treatment with JNJ-26481585. D, human colon carcinoma C170HM2 cells were injected into the peritoneal cavity of male MFI nude mice resulting in growth of liver metastasis. Columns, mean liver tumor weight (g) for each group on day 40; bars, SEM. Mice were treated with JNJ-26481585 from day 7 to day 40 (5 mg/kg once daily i.p.), 25 mg/kg 5-FU/Leucovorin i.v. on days 7, 9, 11, and 13 with the cycle repeated on day 21 and 35, or vehicle control (20% hydroxypropyl-β-cyclodextrin daily i.p.).

Immunohistochemical analysis of the HCT116 colon xenograft tissue revealed a strong induction of pan H3 acetylation in nearly all tumor cells after treatment with JNJ-26481585 (Fig. 6C, top). This induction was not tumor specific, as similar effects were observed in epidermis and also in hair follicle cells. Since we showed that JNJ-26481585 also induced acetylation of histone H4-K16 and trimethylation of histone H4-K20 in tumor cells in vitro, we further investigated this modification, as its loss is a hallmark of human cancer (21). Interestingly, induction of trimethylation of histone H4-K20 seemed to be more specific than pan-H3 induction, as it was observed prominently in tumor tissue, but was much less evident in epidermis or hair follicles (Fig. 6C, middle). Acetylation of histone H4-K16 was observed in tumor tissue and hair follicles, but not in mouse epidermis (Fig. 6C, bottom).

JNJ-26481585 is a pan HDAC inhibitor that also potently induced tubulin acetylation, which has been shown to be involved in tumor cell migration (22). We therefore also tested JNJ-26481585 in a C170HM2 colorectal liver metastasis model. As shown in Fig. 6D, there was a significant 87% reduction in mean liver tumor burden in the JNJ-26481585–treated group (0.380 g reduced to 0.050 g; P = 0.016). JNJ-26481585 inhibitory effect was significantly greater than that observed by 5-FU/Leucovorin, which reduced tumor weight by 60% (0.586 g reduced to 0.234 g; P = 0.045). In summary, these data show that JNJ-26481585 shows high in vivo antitumoral potency in several preclinical colorectal cancer tumor models, and outperforms in this setting the first-generation HDAC inhibitor vorinostat and standard of care 5-FU.

Discussion

HDAC inhibitors in clinical development have shown activity in hematologic malignancies, and vorinostat has been approved for the treatment of cutaneous T-cell lymphoma (6). Thus far, however, the activity of this class of agents has been limited in solid cancer indications as single agents. We hypothesized that current HDAC inhibitors may not impact on solid tumor growth due to their suboptimal potency for class I HDACs and transient pharmacodynamic responses. In preclinical studies, prolonged compound exposure is needed to induce apoptosis in vitro or to inhibit tumor growth in vivo. To identify a novel HDAC inhibitor with superior pharmacodynamic properties, we developed a model allowing fluorescence-based in vivo screening for HDAC1 inhibition in tumors (15). The model was aimed at identifying HDAC1 inhibitors, because downregulation of HDAC1, HDAC2, and HDAC3 drives epithelial tumor cell apoptosis (7). In vivo pharmacodynamic analysis of 140 preselected pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585, a novel second-generation oral pan-HDAC inhibitor. JNJ-26481585 has exquisite potency toward all class I HDAC enzymes (IC50 values of 0.11, 0.33, and 4.8 nmol/L, for HDAC1, HDAC2, and HDAC3, respectively). In agreement with the powerful inhibition of class I HDAC enzymes, JNJ-26481585 showed broad spectrum antiproliferative activity (IC50, 3.1-246 nmol/L) in lung, breast, colon, prostate, brain, and ovarian cancer cell lines at concentrations that are significantly below those achieved in plasma of mice xenografted with human solid tumors (Cmax, 4.9 μmol/L, dosing i.p. at 10 mg/kg for 21 days). JNJ-26481585 potently induced the HDAC1-suppressed p21waf1,cip1 promoter in vivo, and induced a continuous pharmacodynamic response (histone H3 acetylation) in tumor tissue. The potent and prolonged activity of JNJ-26481585 in tissues was found to translate into superior preclinical tumor growth inhibition. JNJ-26481585 completely inhibited the growth of Ras mutant pre-established HCT116 colon xenografts, whereas both 5-FU and the first-generation HDAC inhibitor vorinostat showed only modest activity under these settings. This is in agreement with the short induction of histone H3 and H4 acetylation observed with vorinostat in CWR22 prostate xenografts, i.e., back to baseline levels 12 hours after dosing (23).

Colon cancer is potentially an interesting indication for these agents due to its high frequency of Ras mutations. k-Ras mutant colon cancer cells have been shown to have higher sensitivity to HDAC inhibitors (24), and more recently, Ras mutations were found to sensitize nontransformed intestinal epithelial cells to undergo apoptosis in response to HDAC inhibition (25). In our hands, there was a tendency for JNJ-26481585 to induce greater apoptosis in Ras mutant (HCT116) than Ras wild-type (Colo-205 and HT-29) tumor cells in vitro (Fig. 5C). It is notable that although JNJ-26481585 strongly inhibits tumor growth in vivo of Ras mutant NSCLC xenografts (Supplementary Fig. S9), no efficacy in vivo was observed in Ras wild-type NCI-H1703 NSCLC xenografts (data not shown). Also, in vivo, we found Ras mutant HCT116 xenografts to be significantly more sensitive to HDAC inhibitors than Ras wild-type HT-29 and Colo-205 xenografts (data not shown). Overexpression of class I HDAC family members has been found in primary colon cancer tissue, i.e., of HDAC1 (36%), HDAC2 (58%), and HDAC3 (73%; ref. 26). Overexpression occurs in a subset of strongly proliferating dedifferentiated colorectal carcinoma and is associated with reduced patient survival (26).

The increased expression of HDAC2 has been linked to loss of adenomatous polyposis coli (APC), and has been shown to result in resistance to apoptosis and lower sensitivity to HDAC inhibitor vorinostat (27, 28). In human colon cancer cell lines, in vitro JNJ-26481585 induced potent apoptosis at 3 to 30 nmol/L, both in APC wild-type (HCT116) and in APC mutant (HT-29) backgrounds. It will be of interest to investigate whether the high potency of JNJ-26481585 toward HDAC2 renders it more effective also in an APC-mutated background.

The high potency of JNJ-26481585 toward HDAC1 and HDAC2 is not only of interest in the context of colon cancer. For example, HDAC1 activity is highly upregulated in prostate cancer tissue, due to TMPRSS2 fusions, and these tumors have been suggested to be highly sensitive to HDAC inhibitors (29). Furthermore, inhibiting class I HDACs lies at the basis of the synergy of HDAC inhibitors with DNA-damaging therapies such as chemotherapeutics and radiation (30). HDAC1 was recently also shown to be responsible for sensitization of cells to TRAIL-induced apoptosis (31). Finally, JNJ-26481585 also potently upregulated the HDAC1-suppressed expression of E-cadherin (at concentrations as low as 30 nmol/L), which has been shown to result in sensitization to epidermal growth factor receptor inhibitors in NSCLCs (32).

Although JNJ-26481585 showed a >500 fold higher potency toward HDAC1 than vorinostat, their in vitro potency toward HDAC6 was comparable. In agreement with this observation, in tumor cells, JNJ-26481585 and vorinostat showed equipotent inhibition of deacetylation of HDAC6 substrates tubulin and Hsp90, as evidenced by an increase in tubulin acetylation, induction of Hsp70, and loss of Hsp90 client c-Raf at concentrations as low as 100 nmol/L. Surprisingly, however, the extent of induction of tubulin acetylation is much lower with JNJ-26481585 compared with vorinostat or panobinostat (Fig. 4). HDAC-6 is unique among deacetylases in having two HDAC domains, of which both domains possess α-tubulin deacetylase activity (33). A model in which JNJ-26481585 only targets one domain of HDAC6 may explain the lower maximal extent of tubulin acetylation observed. JNJ-26481585, however, shows all characteristics of a genuine inhibitor of HDAC6. First, we found potent synergy between JNJ-26481585 and bortezomib (10, 34) in a broad panel of hematologic cell lines (data not shown). Second, JNJ-26481585 potently inhibited the growth of colon metastasis in the liver (Fig. 6D), which is in agreement with the proposed role of HDAC6 in tumor cell migration and spreading of metastasis (8, 33, 35-37). Finally, JNJ-26481585 showed strong impairment of the function of Hsp90, also a substrate of HDAC6. It should be noted though that other HDAC family members also regulate Hsp90. The class I–specific HDAC inhibitors MS-275 and depsipeptide disrupt Hsp90 function in an indirect manner, through HDAC1-mediated acetylation and inhibition of Hsp70 function (38, 39). Similarly, Park et al. (40) showed that knockdown of HDAC10 resulted in depletion of Hsp90 client vascular endothelial growth factor receptor.

The distinct biological roles of HDAC family members strongly suggest that the selectivity profile of HDAC inhibitors will have major consequences on their clinical activities. JNJ-26481585 shows a broad spectrum of activity toward both class I and class II HDAC family members. Its favorable pharmacodynamic profile results in potent in vivo antitumoral activity as a single agent in preclinical tumor models. These properties make JNJ-26481585 a promising second-generation HDAC inhibitor with potential clinical applicability in a broad spectrum of human solid and hematologic malignancies. JNJ-26481585 is currently in phase I clinical trials.

Disclosure of Potential Conflicts of Interest

J. Arts: stock owner, Johnson & Johnson. The other authors disclosed no potential conflicts of interest.

Acknowledgments

We thank the chemists Sven van Brandt, Marc Willems, and Leo Backx, and Steve McClue for critical reading of the manuscript.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

    • Received March 4, 2009.
    • Revision received July 22, 2009.
    • Accepted August 10, 2009.

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Clinical Cancer Research: 15 (22)
November 2009
Volume 15, Issue 22

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JNJ-26481585, a Novel “Second-Generation” Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity
Janine Arts, Peter King, Ann Mariën, Wim Floren, Ann Beliën, Lut Janssen, Isabelle Pilatte, Bruno Roux, Laurence Decrane, Ron Gilissen, Ian Hickson, Veronique Vreys, Eugene Cox, Kees Bol, Willem Talloen, Ilse Goris, Luc Andries, Marc Du Jardin, Michel Janicot, Martin Page, Kristof van Emelen and Patrick Angibaud
Clin Cancer Res November 15 2009 (15) (22) 6841-6851; DOI: 10.1158/1078-0432.CCR-09-0547
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