Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical

Published OnlineFirst October 27, 2009; DOI: 10.1158/1078-0432.CCR-09-0547

Cancer Therapy: Preclinical

JNJ-26481585, a Novel “Second-Generation” Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity Janine Arts,1 Peter King,1 Ann Mariën,1 Wim Floren,1 Ann Beliën,1 Lut Janssen,1 Isabelle Pilatte,1 Bruno Roux,1 Laurence Decrane,1 Ron Gilissen,1 Ian Hickson,1 Veronique Vreys,1 Eugene Cox,2 Kees Bol,2 Willem Talloen,3 Ilse Goris,4 Luc Andries,5 Marc Du Jardin,1 Michel Janicot,1 Martin Page,1 Kristof van Emelen,1 and Patrick Angibaud1

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, there- by 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 characteriza- tion 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)

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, HDAC inhibitors are in clinical development where activity terminal differentiation, and apoptosis in a broad spectrum of has been observed mainly in hematologic malignancies. human tumor cell lines in vitro, and have antiangiogenic and This has led to the recent approval of vorinostat (suberoylani- antitumor activity in human xenograft models (1–5). Several lide hydroxamic acid) for the treatment of cutaneous T-cell lymphoma (6). Four classes of HDACs have been identified: classes I, II, III, 1 Authors' Affiliations: Ortho Biotech Oncology Research and Development, and IV. Most HDAC inhibitors under clinical development act 2Clinical Pharmacology, 3Biostatistics, and 4Functional Genomics, Johnson & Johnson Pharmaceutical R. & D., Beerse, Belgium; and 5HistoGenex, on three distinct classes of HDAC enzymes: class I, comprising Edegem, Belgium HDAC1-3 and HDAC8; class IIa, comprising HDAC4, HDAC5, Received 3/4/09; revised 7/22/09; accepted 8/10/09; published OnlineFirst 10/27/09. HDAC7, HDAC9; and class IIb, comprising HDAC6 and The costs of publication of this article were defrayed in part by the payment HDAC10. HDAC11 is the sole member of class IV HDACs. In- of page charges. This article must therefore be hereby marked advertisement hibition of class I HDACs results in the acetylation of nuclear 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 histone proteins, which affects tertiary chromatin structure and Research Online (http://clincancerres.aacrjournals.org/). leads to altered expression of genes involved in cell prolifera- Requests for reprints: Janine Arts, Oncology Research and Early Develop- tion, apoptosis, and differentiation. Class I HDAC activity is ment, Johnson & Johnson Pharmaceutical Research and Development, key for uncontrolled proliferation of cancer cells, because Turnhoutseweg 30, 2340 Beerse, Belgium. Phone: 0032-14606325; Fax: 0032-14605403; E-mail: [email protected]. downregulation of HDAC1 and HDAC3 expression results in F 2009 American Association for Cancer Research. increased histone acetylation and inhibition of tumor cell pro- doi:10.1158/1078-0432.CCR-09-0547 liferation (7). In contrast to the class I HDAC family members,

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Cancer Therapy: Preclinical

analysis of 140 potent pyrimidyl-hydroxamic acid analogues re- Translational Relevance sulted in the identification of JNJ-26481585, which after once daily oral administration induced continuous histone H3 acet- Histone deacetylase (HDAC) inhibitors have ylation and complete tumor growth inhibition in human shown clinical activity in hematologic malignan- HCT116 colon tumors. cies, but their activity in solid tumor indications The potent antitumoral activity as a single agent in preclinical has been somewhat limited. We hypothesized that models combined with its favorable pharmacodynamic profile this could be related to the transient pharmacody- makes JNJ-26481585 a promising second-generation HDAC in- “ ” namic effects of first-generation HDAC inhibitors hibitor. The compound is currently in clinical studies, to eval- in tumor cells. This hypothesis is further supported uate its potential applicability in a broad spectrum of human by the observation that HDAC inhibitors need to be solid and hematologic malignancies. given chronically and prolonged exposure to HDAC inhibitors seems essential to achieve effective tumor cell death. Here, we identified JNJ-26481585, Materials and Methods a “second-generation” HDAC inhibitor with prolonged pharmacodynamic response in vivo.In Compounds. JNJ-26481585, R306465 and vorinostat (Fig. 1D), pa- agreement with the hypothesis, JNJ-26481585 nobinostat, CRA-024781, and mocetinostat were synthesized according showed superior efficacy compared with both stan- to published methods. All compounds were dissolved in DMSO as 5 dard of care agents and first-generation HDAC inhi- mmol/L stock solutions and kept at room temperature. HDAC activity assays. Recombinant HDAC activity assays were bitors in preclinical tumor models. These studies done by Reaction Biology Corporation. In all cases, full-length HDAC suggest that an HDAC inhibitor with continuous proteins were expressed using baculovirus-infected Sf9 cells. In addi- pharmacodynamic activity may show activity in tion, HDAC3 was coexpressed as a complex with human NCOR2. For solid tumor malignancies. JNJ-26481585 is current- assessing activity of HDAC1-containing cellular complexes, immuno- ly being evaluated in the clinic in tumors known to precipitated HDAC1 complexes were incubated with an [3H]acetyl- be driven by high class I HDAC activity. labeled fragment of histone H4 peptide [biotin-(6-aminohexanoic) 3 Gly-Ala-(acetyl[ H])Lys-Arg-His-Arg-Lys-Val-NH2; Amersham Pharma- cia Biotech] as described (12). Equal amounts of HDAC1 were immunoprecipitated as indicated by Western blot analysis. HDAC1 activity class II HDACs are not as widely involved in processes that con- results are presented as mean ± SD of three independent experiments trol survival of solid cancer cells. Downregulation of class IIa on a single lysate. members HDAC4 and HDAC7 in HeLa cells using siRNA tech- Cell proliferation and apoptosis assays. All cell lines were obtained nology did not result in decreased proliferation (7), and inhi- from American Type Culture Collection and cultured according to in- bition of the class IIb enzyme HDAC6 similarly did not widely structions. The effect of HDAC inhibitors on cell proliferation was mea- affect tumor cell survival. HDAC6, however, is a deacetylase for sured using an MTT as described (12). Proliferation of non–small cell – tubulin and Hsp90. HDAC6 inhibitors decrease cell motility lung carcinoma (NSCLC) cell lines was assessed using an Alamar Blue (8, 9) and deplete oncogenic Hsp90 client proteins, resulting based assay as described (12). For proliferation of hematologic cell lines, cells were incubated for 72 h and the cytotoxic activity was eval- in potentiation of therapeutics such as paclitaxel, trastuzumab, uated by MTSassay. Data are presented as mean IC 50 or IC40 ±SDofat and bortezomib (10, 11). least three independent experiments. For apoptosis assays, human tu- We previously identified R306465, a highly potent class I se- mor cells were incubated for 24, 48, and 96 h with JNJ-26481585 at the lective HDAC inhibitor, showing oral antitumor activity in hu- indicated concentrations. Cells were stained for Annexin V and 7-AAD, man tumor–bearing mice (12). Although R306465 showed according to the manufacturer's instructions and analyzed (Guava PCA- antitumoral activity in nonestablished tumor models in vivo, 96 Nexin kit, Guava Technologies). The number of apoptotic and ne- its activity was found to be modest when treating immunode- crotic cells was expressed as a percentage of the total number of cells ficient mice carrying pre-established tumors. Pharmacodynamic present in the well. Total cell number was expressed as a percentage studies revealed that R306465 has a short half-life and only of control, and the percentage of apoptotic/necrotic cells present in transiently induces histone acetylation in vivo, a feature shared the absence of compound was subtracted from all values. All results shown are an average of three independent experiments (± SD). with a number of other HDAC inhibitors in clinical develop- Western blot analysis. Human A2780 ovarian carcinoma cells were ment. Vorinostat, for example, has an apparent plasma half-life incubated with the indicated concentrations of JNJ-26481585, CRA- of 91 to 127 minutes and the induction of H3 acetylation in 024781, and mocetinostat. Total cell lysates were prepared using hot circulating peripheral blood mononuclear cells was found to lysis buffer containing 1% SDS in 10 mmol/L Tris (pH 7.4), supple- be transient (13, 14). mented with 1 mmol/L sodium orthovanadate. Cleared cell lysates To identify a “second-generation” HDAC inhibitor with im- were fractionated by SDS-PAGE and transferred to polyvinylidene proved single agent antitumoral efficacy, we aimed to design a difluoride membranes (Bio-Rad Laboratories). Levels of acetylated molecule with high potency toward class I HDACs, showing a H3 (AcH3) and H4 histones (Upstate/Millipore), histone H4-acetyl in vivo K16 (Abcam), histone H4-trimethyl K20 (Abcam), total H3 protein prolonged pharmacodynamic response . We, therefore, waf1,cip1 developed an experimental tumor model which allows nonin- (Abcam), E-Cadherin (Abcam), p21 (BD Transduction Laborato- ries), total tubulin (Sigma), acetylated tubulin (Sigma), Hsp70 (Stress- vasive real-time analysis of HDAC1 activity in tumor tissue gen), and c-Raf (BD Transduction Laboratories) protein were detected (15). In this model, human A2780 ovarian carcinoma cells using the appropriate antibodies. To control for equal loading, blots have been engineered with a reporter gene construct encoding were stripped and reprobed with anti-actin (Oncogene Research Pro- the fluorescent ZsGreen protein, whose expression is under the ducts) or anti-lamin B1 (Zymed, Invitrogen) antibodies. As secondary waf1,cip1 control of the p21 promoter, a gene constitutively re- antibodies, horseradish peroxidase–labeled anti-mouse (Santa Cruz pressed by the HDAC1 protein (16). In vivo pharmacodynamic Biotechnology), horseradish peroxidase–labeled anti-rabbit (Zymed),

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JNJ-26481585: Novel “Second-Generation” HDAC Inhibitor

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.

fluorochrome-labeled anti-mouse (Rockland), or fluorochrome-labeled significant. To generate the xenograft model allowing imaging of waf1,cip1 anti-rabbit (Invitrogen Molecular Probes) were used. Protein-antibody HDAC1 activity, an HDAC1-responsive p21 promoter construct, waf1,cip1 complexes were then visualized by chemiluminescence (Pierce Chemi- the -1300 to +88 region of the p21 promoter, was cloned into cal Co.) or fluorescence (Odyssey, Li-Cor Biosciences) according to pGL3-basic-ZsGreen and stably transfected into A2780 ovarian carcino- waf1,cip1 manufacturer's instructions. ma cells (15). For in vivo analysis, mice carrying A2780-p21 Zs- In vivo xenograft studies. JNJ-26481585, R306465, and vorinostat Green tumors were treated from day 12 after grafting both orally were formulated at 2 mg/mL in 20% hydroxypropyl-β-cyclodextrin (p.o.) and i.p. once daily for 4 d with solvent or the indicated dose (final pH 8.7). 5-Fluorouracil (5-FU) was formulated in 20% hydroxy- of HDAC inhibitors. Tumors were evaluated for fluorescence by an propyl-β-cyclodextrin (pH 4.4). All mice used in the in vivo studies were automated Whole-Body Imaging system (12, 15). treated according to the ethical guidelines prescribed by United King- Immunohistochemistry. HCT116 colon xenograft tissue and mouse dom Coordinating Committee on Cancer Research. HCT116 human skin were embedded in paraffin and further processed for immunoflu- colon carcinoma cells were injected s.c. (107 cells/200 μL) into the orescence staining. The following primary antibodies were used: acety- inguinal region of athymic male NMRI nu/nu mice purchased from Jan- lated histone H3 (Upstate/Millipore), histone H4-acetyl K16 (Abcam), vier. Tumor size was determined using caliper measurement and tumor or histone H4-trimethyl K20 (Abcam). After overnight incubation with volume was determined by using the formula: TV =(a2 × b)/2 (in which the primary antibody, an appropriate goat-specific CY3-conjugated sec- a represents the width and b the length). The human colon carcinoma ondary antibody (Jackson Immunoresearch Laboratories) was used. For cell line C170HM2 was derived originally from a primary tumor. negative controls, the primary antibody was omitted. For vessel locali- C170HM2 cell suspensions (1.5 × 106 cells) were injected into the peri- zation, tumors were collected using transcardial perfusion fixation with toneal cavity of male MFI nude mice (bred within the Cancer Studies 4% paraformaldehyde. After cryoprotection, cryosections of 10-μm Unit at the University of Nottingham, United Kingdom) in 1 mL thickness were made and mounted on glass slides. The xenografts were of 0.9% sodium chloride (pH 7.3). The liver was exposed, excised, stained for actin using BODIPY 558/568-phalloidin (Invitrogen Molec- and analyzed as described (17). Statistical analysis was done using ular Probes) and the vessels were stained using CD-31 antibody (BD Wilcoxon Mann-Whitney analysis with P < 0.05 considered statistically Transduction Laboratories) and a goat anti-rat secondary antibody

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Cancer Therapy: Preclinical coupled to Cy3 (Jackson Immunoreasearch Laboratories). Images were erated doses of JNJ-26481585 and vorinostat were determined, taken using a Zeiss Axioplan 2 microscope equipped with an Axiocam after a 20-day dosing period, as the doses that resulted in <15% HRm camera and Axiovision imaging software (Zeiss). weight loss and no lethality (data not shown). In JNJ- In vivo pharmacokinetic and pharmacodynamic analysis. Tumor ma- 26481585–treated tumors, fluorescence was not uniformly dis- terial from mouse xenografts was isolated and homogenized using son- tributed throughout the tissue (Fig. 1B and C). Generally, focal ication. H3 acetylation was determined by ELISA or MSD (Meso Scale Discovery), according to the manufacturer's instructions. Briefly, the anti- spots of high fluorescence were surrounded by areas with weak- histone PAN antibody (Chemicon/Millipore) was spotted onto the er intensity. The areas of these high intensity spots contained base of a 96-well MSD Standard Bind plate. After blocking, the xeno- from a few to hundreds of cells and did not colocalize with graft sample [2.5 μg/25 μL 10 mmol/L Tris (pH 7.4) supplemented tumor vasculature (Fig. 1B). There was no difference in fluores- with 1 mmol/L sodium orthovanadate] or calibrator sample (Upstate/ cence between the peripheral and central parts of the tumor, Millipore) was added and the array was incubated with anti-histone H3 indicating exposure of the drug and/or a biological response antibody (Upstate/Millipore). Assay results were read using the MSD throughout the entire tumor (data not shown). Sector Imager 6000. To investigate the reason for the exquisite potency of JNJ- waf1, cip1 The model-based analysis of the pharmacokinetic and pharmacody- 26481585 in activating the HDAC1-regulated p21 namic data were done using S-PLUS version 7.0 (Insightful Corpora- promoter in vivo, we investigated inhibition of HDAC1 enzyme tion) and NONMEM version 5 (ICON). The time course of drug concentration in the plasma over the duration of the study was de- activity by immunoprecipitating HDAC1 complexes from in vitro scribed on the basis of a two-compartment disposition model. This A2780 ovarian carcinoma cells . JNJ-26481585 inhibited concentration versus time profile can be described by the following HDAC1 complexes with an IC50 value of 0.16 ± 0.02 nmol/L -β˙t n equation: [Ct = I* (A ˙ e ˙ + B ˙ e ) eq. PKPD1]. Ct is the concentration (average ± SD, = 3), which is 20-fold more potent than t I of JNJ-26481585 in the plasma at time since the first dose, is the R306465 (IC50, 3.31 ± 0.78 nmol/L) and 530-fold more potent administration schedule of the compound (10 mg/kg every 24 h for than vorinostat (IC50,85±16nmol/L).HDAC1suppresses waf1,cip1 21 d), * denotes convolution, whereas A and B, and α and β, are the p21 gene expression by deacetylating nuclear histone coefficients and the exponents describing the disposition function of proteins, which affects tertiary chromatin structure (16). We, JNJ-26481585, respectively. The time course of AcH3 response in the therefore, measured the kinetics of histone H3 acetylation in tumor at day 1 and day 7 was described on the basis of an indirect re- the A2780 xenograft tissue in vivo, to assess the impact of the sponse pharmacodynamic model (18). In the model, the level of H3 acetylation at baseline is the net effect of histone acetylation and dea- high HDAC1 potency on the duration of the pharmacodynamic cetylation. Treatment with JNJ-26481585 disrupts this balance by inhi- response. Immunodeficient mice carrying A2780 ovarian tu- bition of deacetylation. The net effect is an increased level of H3 mors were treated once daily with JNJ-26481585 (10 mg/kg, acetylation. The process is described by the following equation: i.p.) and H3 acetylation in the xenograft was analyzed using a quantitative ELISA. As shown in Fig. 2, JNJ-26481585 induces dAcH C 3 ¼ K þ K AcH − JNJ;t potent tumor H3 acetylation (both after the first dose and at dt HAC HDAC ˙ 3;t ˙ 1 IC þ C 50 JNJ;t day 7) and the time course of this drug effect is delayed when KHAC is the presumed zero-order rate constant of histone acetylation compared with the time course of JNJ-26481585 exposure in and KHDAC is the first-order rate constant of histone deacetylation in the plasma: the maximum AcH3 response is observed 2 to the absence of JNJ-26481585. AcH3,t is the concentration of acetylated 5 hours postdose, whereas the maximum exposure in plasma t, C histone on the tumor tissue at time JNJ,t is the plasma concentration is observed at 30 minutes postdose. Importantly, after dosing t IC of JNJ-26481585 at time , and 50 is the plasma concentration of JNJ- JNJ-26481585 for 7 days, there is a higher basal level of H3 26481585 resulting in 50% inhibition of rate of histone deacetylation. acetylation in the tumor than on day 1. This indicates that the AcH3 response further accumulates between day 1 and Results day 7, and H3 acetylation in the tumor tissue is continuously induced, although drug plasma exposure does not show accu- Identification of JNJ-26481585 as a potent HDAC1 inhibitor mulation between day 1 and day 7. Tumor concentrations of in vivo. To identify an HDAC inhibitor with improved poten- JNJ-26481585 were measured on days 14 and 21 and show that cy and prolonged pharmacodynamic response in tumor tissue, there is continuous exposure of HDAC1 to JNJ-26481585 in tu- we preselected 140 pyrimidyl-hydroxamic acid analogues mor tissue (Supplementary Fig. S7). The short plasma half-life that were all potent HDAC inhibitors in HeLa nuclear extracts of JNJ-26481585 in nude mice is in agreement with the in vitro (IC50, 0.05-100 nmol/L). We subsequently analyzed all observed extensive rodent-specific first-pass metabolism. There- these promising compounds in vivo in an HDAC1-responsive fore, subsequent antitumor studies with JNJ-26481585 in nude A2780 ovarian tumor screening model. In this model, the mice were done i.p., the optimal route of administration for waf1,cip1 HDAC1-regulated p21 promoter controls a fluorescent this compound in rodents. ZsGreen protein. We previously showed that induction of tu- The time course of AcH3 response in the tumor at day 1 and mor fluorescence fully predicts tumor growth inhibition after day 7 could be described on the basis of an indirect response dosing experimental agents for 3 days only (15). As shown in pharmacodynamic model (18). In this model, the drug effect Fig. 1A, JNJ-26481585 was the only compound identified that (inhibition of the deacetylation of histone) is integrated with induced a bright and intense fluorescence in the tumor xeno- the rates of both histone acetylation and deacetylation (see Ma- grafts after dosing for 3 days at its maximal tolerated dose terials and Methods section). Figure 2 shows that AcH3 re- (10 mg/kg i.p. and 40 mg/kg p.o.). The bright fluorescence sponse continues to increase with dosing of JNJ-26481585; was in contrast to the more weak induction observed with other then is expected to return to pretreatment values ∼21 days after clinical HDAC inhibitors such as R306465 (40 mg/kg, oral; treatment termination. KHDAC, the first-order rate constant of ref. 12) and vorinostat (200 mg/kg, p.o.), which were also histone deacetylation in the absence of JNJ-26481585, was administered at their maximal tolerated dose. The maximal tol- found to be highly different between different tumors, being

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JNJ-26481585: Novel “Second-Generation” HDAC Inhibitor

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).

19 U/h in A2780 ovarian xenografts, but much lower (1.8 U/h) studied inhibition by analyzing HDAC substrate acetylation in in HCT116 colon xenografts (data not shown). The plasma A2780 ovarian carcinoma cells. JNJ-26481585 induced H3 and concentration of JNJ-26481585 resulting in 50% inhibition of H4 acetylation at concentrations as low as 30 to 100 nmol/L rate of histone deacetylation (IC50), however, was comparable (Fig. 4), indicating high potency toward class I HDACs in tumor across tumors, being 5.9 and 7.9 ng/mL in A2780 ovarian and cells. JNJ-26481585 also induced acetylation and trimethyla- HCT116 colon xenografts, respectively (14 and 18 nmol/L). tion at Lys16 and Lys20 of histone H4, respectively (Fig. 4). These data indicate that JNJ-26481585 shows comparable po- Loss of these modifications is a common hallmark of human tency across tumor types, independent of their basal level of cancer (21). Genes specifically silenced by HDAC1, such as waf1,cip1 HDAC activity. p21 and E-cadherin, were induced with similar high po- The observation that JNJ-26481585 causes prolonged H3 tency, confirming potent HDAC1 inhibition in tumor cells. Sur- acetylation in the tumor tissue, despite its short plasma half- prisingly, despite modest in vitro potency against recombinant life, is in contrast to the effects of less potent HDAC1 inhibitors. HDAC6, in A2780 tumor cells, JNJ-26481585 also showed high For example, R306465 was previously found to cause only a cellular potency toward HDAC6. JNJ-26481585 induced acety- short transient induction of H3 acetylation in tumor tissue at lation of HDAC6 substrate tubulin at concentrations as low as 1 and 4 hours postdose, returning to baseline levels at 8 hours 30 to 100 nmol/L. Similarly, Hsp90 activity is inhibited, as (data not shown). evidenced by induction of Hsp70 and loss of Hsp90 client HDAC isotype specificity of JNJ-26481585. To characterize c-Raf. Therefore, in tumor cells, JNJ-26481585 shows equipo- the inhibition of other HDAC family members by JNJ- tent inhibition of HDAC1 and HDAC6 and behaves like a true 26481585, we tested its potency toward a broad panel of pan-HDAC inhibitor. recombinant HDAC enzymes in vitro.AsshowninTable1, We further compared the specificity profile of JNJ-26481585 JNJ-26481585 shows activity toward all HDAC enzymes tested with other HDAC inhibitors in clinical development (Fig. 4). with highest potency in vitro observed toward recombinant Vorinostat has been shown to induce histone H3 acetylation HDAC1 (IC50, 0.11 ± 0.03 nmol/L), which is comparable with only at 1 to 3 μmol/L, whereas tubulin acetylation and the potency observed toward HDAC1-immunoprecipitated Hsp70 induction were already evident at 100 nmol/L, indicat- complexes from tumor cells (IC50, 0.16 ± 0.02 nmol/L). Lowest ing a 10-fold higher potency for HDAC6 for this agent (12). in vitro potency was observed toward HDAC6, 7 and 9 (IC50, This is in line with the higher relative potency for HDAC6 with 32.1-119 nmol/L). As shown in Fig. 3A and B, unsupervised vorinostat compared with JNJ-26481585 observed on these en- clustering of isotype specificity with a panel of other HDAC in- zymes in vitro. Also, class I–specific benzamide mocetinostat inhibitors in clinical development revealed that in vitro JNJ- duced histone H3 acetylation at 3 μmol/L, but did not affect 26481585 and its predecessor R306465 show a class I–specific HDAC6 substrates. Finally, in agreement with previous publica- inhibition profile that most resembles that of the fully class – tions, the hydroxamic acid–based HDAC inhibitors CRA- specific benzamide mocetinostat (r = 0.75; ref. 19), with the 024781 and panobinostat (12) both also inhibited HDAC1/ distinction that JNJ-26481585 is 100-fold more potent than HDAC6 at similar concentrations, because both substrates were mocetinostat. JNJ-26481585 is most distinct from vorinostat acetylated at 100 and 300 nmol/L (10). (r = 0.26) and CRA-024781 (r = 0.40; ref. 20). JNJ-26481585 has broad spectrum antiproliferative activity Analyzing specificity profiles based on inhibition of isolated against solid and hematologic cancer cell lines and induces recombinant enzymes can be misleading, especially for HDACs, apoptosis. Class I HDACs such as HDAC1 have been shown which function as large multiprotein complexes. We, therefore, to be key for tumor cell proliferation (7). As JNJ-26481585

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Cancer Therapy: Preclinical

Table 1. Activity toward tested HDAC enzymes (nM ± SD)

HDAC JNJ-26481585 R306465 Vorinostat Panobinostat Mocetinostat CRA-024781 Class

1 0.11 ± 0.03 2.54 ± 0.3661.8± 10.4 0.31 ± 0.05 9.26±1.18 21 ± 1.77 I 2 0.33 ± 0.068.76±2.94 241 ± 17 1.51 ± 0.20 35.6±13.0 16.6I 63.4± 3 4.86 ± 0.69 9.04 ± 2.94 257 ± 83 1.69 ± 0.20 192 ± 39.9 148 ± 16.9 I 8 4.26± 2.97 27.4 ± 13.1 212 ± 20.0 38.9 ± 10.0 Inactive 370 ± 84.4 I 4 0.64 ± 0.18 4.64 ± 0.35 154 ± 33 0.42 ± 0.08 810 ± 262 60.4 ± 6.92 IIa 5 3.69 ± 0.63 7.15 ± 2.32 101 ± 40 0.84 ± 0.17 899 ± 186 48.2 ± 18.0 IIa 7 119 ± 3.61 45.9 ± 8.20 2488 ± 881 38.6 ± 13.2 Inactive 350 ± 97.9 IIa 9 32.1 ± 4.77 28.2 ± 1.11 182 ± 46.8 1.65 ± 0.14 Inactive 168 ± 50.1 IIa 6 76.8 ± 6.35 66.0 ± 3.92 20.0 ± 3.24 1.98 ± 0.28 Inactive 11.5 ± 1.98 IIb 10 0.46± 0.07 4.34 ± 1.14 152 ± 30.8 0.61± 0.12 5799 ± 2200 52.0 ± 8.18 IIb 11 0.37 ± 0.08 2.10 ± 0.58 100 ± 39.5 0.32 ± 0.03 195 ± 101 13.6± 3.04 IV

shows such high potency in tumor cells versus class I HDACs, ing analysis was done. As shown in Fig. 5C, after 48 hours of we, therefore, investigated the antiproliferative effects of JNJ- incubation, in all cell lines investigated, JNJ-26481585 treat- 26481585 in a broad panel of human tumor cell lines from ment at 3 to 300 nmol/L caused a significant increase in the both solid and hematologic origin. As indicated in Fig. 5A, percentage of cells positive for Annexin V in a concentration- JNJ-26481585 inhibited cell proliferation in all lung, breast, co- dependent manner indicative of apoptosis. An increase in the lon, prostate, brain, and ovarian tumor cell lines tested, with number of necrotic cells was also observed, which likely repre- IC50 values ranging from 3.1 to 246 nmol/L. We also observed sents apoptotic cells in later stages of cell death. that JNJ-26481585 exhibited a more potent antiproliferative ef- Antitumor efficacy of JNJ-26481585. As JNJ-26481585 po- fect than vorinostat, R306465, panobinostat, CRA-24781, or tently induces tumor cell apoptosis, and shows a continuous mocetinostat in various human cancer cell lines tested (Supple- pharmacodynamic response in tumor tissue in vivo, we subse- mentary Fig. S8). This is in agreement with its high relative po- quently investigated whether these properties indeed translated tency toward class I HDACs (HDAC1/2) compared with other into improved efficacy in preclinical tumor in vivo models. As HDAC inhibitors (Fig. 3). shown in Fig. 6A, JNJ-26481585 administered continuously This antiproliferative effect was not dependent on p53 geno- for 14 days (once daily, 10 mg/kg i.p.) strongly inhibited the typic status, or Ras mutational status in colon and lung tumor growth of large pre-established HCT116 colon xenografts cells (data not shown). Similarly, as shown in Fig. 5B, JNJ- (320 ± 10 mm3 at start of treatment). At the end of the study, 26481585 inhibited the proliferation with comparable potency JNJ-26481585 inhibited tumor volume by 76% (treated versus of acute lymphoblastic leukemia, acute myelogenous leukemia, control = 24), which is superior to the activity of the clinical chronic lymphocytic leukemia, chronic myelogenous leukemia, standard of care agent 5-FU (41% inhibition). The first-genera- lymphoma, and myeloma tumor cells (IC40, 4.5-166 nmol/L). tion HDAC inhibitor vorinostat showed only modest activity in To investigate whether the antiproliferative effect of JNJ- the model (26% inhibition). In agreement with its low antitu- 26481585 in A2780 ovarian tumor cells was due to induction mor potency, vorinostat only slightly increased H3 acetylation of cell cycle arrest or cell death, fluorescence-activated cell sort- levels at 4 hours postdose (0.03 ± 0.02 ng/μg protein), whereas

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.

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JNJ-26481585: Novel “Second-Generation” HDAC Inhibitor

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 waf1, cip1 K20 (H4-K20Me 3); total levels of histone H3, tubulin, p21 , 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).

JNJ-26481585 showed a more potent effect (0.22 ± 0.07 ng/μg JNJ-26481585 shows high in vivo antitumoral potency in several protein; data not shown). A subsequent dose-response study to preclinical colorectal cancer tumor models, and outperforms in further explore the potency of JNJ-26481585 showed similar tu- this setting the first-generation HDAC inhibitor vorinostat and mor growth inhibition at 5 mg/kg compared with 20 mg/kg standard of care 5-FU. (87% and 93% inhibition, respectively, Fig. 6B), whereas half-maximal inhibition was obtained at the low dose of 2.5 Discussion mg/kg in the pre-established setting (69% inhibition). Immunohistochemical analysis of the HCT116 colon xeno- HDAC inhibitors in clinical development have shown activ- graft tissue revealed a strong induction of pan H3 acetylation ity in hematologic malignancies, and vorinostat has been ap- in nearly all tumor cells after treatment with JNJ-26481585 proved for the treatment of cutaneous T-cell lymphoma (6). (Fig. 6C, top). This induction was not tumor specific, as similar Thus far, however, the activity of this class of agents has been effects were observed in epidermis and also in hair follicle cells. limited in solid cancer indications as single agents. We hypoth- Since we showed that JNJ-26481585 also induced acetylation of esized that current HDAC inhibitors may not impact on solid histone H4-K16 and trimethylation of histone H4-K20 in tu- tumor growth due to their suboptimal potency for class I mor cells in vitro, we further investigated this modification, as HDACs and transient pharmacodynamic responses. In preclin- its loss is a hallmark of human cancer (21). Interestingly, induc- ical studies, prolonged compound exposure is needed to induce tion of trimethylation of histone H4-K20 seemed to be more apoptosis in vitro or to inhibit tumor growth in vivo. To identify specific than pan-H3 induction, as it was observed prominently a novel HDAC inhibitor with superior pharmacodynamic prop- in tumor tissue, but was much less evident in epidermis or hair erties, we developed a model allowing fluorescence-based follicles (Fig. 6C, middle). Acetylation of histone H4-K16 was in vivo screening for HDAC1 inhibition in tumors (15). The observed in tumor tissue and hair follicles, but not in mouse model was aimed at identifying HDAC1 inhibitors, because epidermis (Fig. 6C, bottom). downregulation of HDAC1, HDAC2, and HDAC3 drives epi- JNJ-26481585 is a pan HDAC inhibitor that also potently in- thelial tumor cell apoptosis (7). In vivo pharmacodynamic anal- duced tubulin acetylation, which has been shown to be in- ysis of 140 preselected pyrimidyl-hydroxamic acid analogues volved in tumor cell migration (22). We therefore also tested resulted in the identification of JNJ-26481585, a novel sec- JNJ-26481585 in a C170HM2 colorectal liver metastasis model. ond-generation oral pan-HDAC inhibitor. JNJ-26481585 has As shown in Fig. 6D, there was a significant 87% reduction in exquisite potency toward all class I HDAC enzymes (IC50 values mean liver tumor burden in the JNJ-26481585–treated group of 0.11, 0.33, and 4.8 nmol/L, for HDAC1, HDAC2, and (0.380 g reduced to 0.050 g; P = 0.016). JNJ-26481585 inhibi- HDAC3, respectively). In agreement with the powerful inhibi- tory effect was significantly greater than that observed by 5-FU/ tion of class I HDAC enzymes, JNJ-26481585 showed broad Leucovorin, which reduced tumor weight by 60% (0.586 g re- spectrum antiproliferative activity (IC50, 3.1-246 nmol/L) in duced to 0.234 g; P = 0.045). In summary, these data show that lung, breast, colon, prostate, brain, and ovarian cancer cell lines

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Cancer Therapy: Preclinical

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. at concentrations that are significantly below those achieved Colon cancer is potentially an interesting indication for these in plasma of mice xenografted with human solid tumors agents due to its high frequency of Ras mutations. k-Ras mutant (Cmax,4.9μmol/L, dosing i.p. at 10 mg/kg for 21 days). colon cancer cells have been shown to have higher sensitivity to JNJ-26481585 potently induced the HDAC1-suppressed HDAC inhibitors (24), and more recently, Ras mutations were waf1,cip1 p21 promoter in vivo, and induced a continuous pharma- found to sensitize nontransformed intestinal epithelial cells to codynamic response (histone H3 acetylation) in tumor tissue. undergo apoptosis in response to HDAC inhibition (25). In our The potent and prolonged activity of JNJ-26481585 in tissues hands, there was a tendency for JNJ-26481585 to induce greater was found to translate into superior preclinical tumor growth apoptosis in Ras mutant (HCT116) than Ras wild-type (Colo- inhibition. JNJ-26481585 completely inhibited the growth of 205 and HT-29) tumor cells in vitro (Fig. 5C). It is notable that Ras mutant pre-established HCT116 colon xenografts, whereas although JNJ-26481585 strongly inhibits tumor growth in vivo both 5-FU and the first-generation HDAC inhibitor vorinostat of Ras mutant NSCLC xenografts (Supplementary Fig. S9), no showed only modest activity under these settings. This is in efficacy in vivo was observed in Ras wild-type NCI-H1703 agreement with the short induction of histone H3 and H4 acet- NSCLC xenografts (data not shown). Also, in vivo,wefound ylation observed with vorinostat in CWR22 prostate xenografts, Ras mutant HCT116 xenografts to be significantly more sensi- i.e., back to baseline levels 12 hours after dosing (23). tive to HDAC inhibitors than Ras wild-type HT-29 and

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JNJ-26481585: Novel “Second-Generation” HDAC Inhibitor

Colo-205 xenografts (data not shown). Overexpression of class For example, HDAC1 activity is highly upregulated in prostate I HDAC family members has been found in primary colon can- cancer tissue, due to TMPRSS2 fusions, and these tumors have cer tissue, i.e., of HDAC1 (36%), HDAC2 (58%), and HDAC3 been suggested to be highly sensitive to HDAC inhibitors (29). (73%; ref. 26). Overexpression occurs in a subset of strongly Furthermore, inhibiting class I HDACs lies at the basis of the proliferating dedifferentiated colorectal carcinoma and is asso- synergy of HDAC inhibitors with DNA-damaging therapies ciated with reduced patient survival (26). such as chemotherapeutics and radiation (30). HDAC1 was re- The increased expression of HDAC2 has been linked to loss cently also shown to be responsible for sensitization of cells to of adenomatous polyposis coli (APC), and has been shown to TRAIL-induced apoptosis (31). Finally, JNJ-26481585 also result in resistance to apoptosis and lower sensitivity to HDAC potently upregulated the HDAC1-suppressed expression of inhibitor vorinostat (27, 28). In human colon cancer cell E-cadherin (at concentrations as low as 30 nmol/L), which lines, in vitro JNJ-26481585 induced potent apoptosis at 3 to has been shown to result in sensitization to epidermal growth 30 nmol/L, both in APC wild-type (HCT116) and in APC mu- factor receptor inhibitors in NSCLCs (32). tant (HT-29) backgrounds. It will be of interest to investigate Although JNJ-26481585 showed a >500 fold higher potency whether the high potency of JNJ-26481585 toward HDAC2 ren- toward HDAC1 than vorinostat, their in vitro potency toward ders it more effective also in an APC-mutated background. HDAC6 was comparable. In agreement with this observation, The high potency of JNJ-26481585 toward HDAC1 and in tumor cells, JNJ-26481585 and vorinostat showed equipo- HDAC2 is not only of interest in the context of colon cancer. tent inhibition of deacetylation of HDAC6 substrates tubulin

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.; ), vorinostat (100 mg/kg twice daily p.o.; ), 5-FU (100 mg/kg once weekly i.p.; ), 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 ( ), 5 mg/kg ( ), 10 mg/kg ( ), or 20 mg/kg ( ) for 21 d. C, H3 acetylation (H3 Ac), histone H4-trimethyl K20 (H4-K20Me 3), 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.).

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Cancer Therapy: Preclinical and Hsp90, as evidenced by an increase in tubulin acetylation, ly, Park et al. (40) showed that knockdown of HDAC10 re- induction of Hsp70, and loss of Hsp90 client c-Raf at concen- sulted in depletion of Hsp90 client vascular endothelial trations as low as 100 nmol/L. Surprisingly, however, the extent growth factor receptor. of induction of tubulin acetylation is much lower with JNJ- The distinct biological roles of HDAC family members 26481585 compared with vorinostat or panobinostat (Fig. 4). strongly suggest that the selectivity profile of HDAC inhibitors HDAC-6 is unique among deacetylases in having two HDAC will have major consequences on their clinical activities. JNJ- domains, of which both domains possess α-tubulin deacetylase 26481585 shows a broad spectrum of activity toward both class activity (33). A model in which JNJ-26481585 only targets one I and class II HDAC family members. Its favorable pharmaco- domain of HDAC6 may explain the lower maximal extent of dynamic profile results in potent in vivo antitumoral activity as a tubulin acetylation observed. JNJ-26481585, however, shows single agent in preclinical tumor models. These properties make all characteristics of a genuine inhibitor of HDAC6. First, we JNJ-26481585 a promising second-generation HDAC inhibitor found potent synergy between JNJ-26481585 and bortezomib with potential clinical applicability in a broad spectrum of hu- (10, 34) in a broad panel of hematologic cell lines (data not man solid and hematologic malignancies. JNJ-26481585 is cur- shown). Second, JNJ-26481585 potently inhibited the growth rently in phase I clinical trials. of colon metastasis in the liver (Fig. 6D), which is in agreement with the proposed role of HDAC6 in tumor cell migration and Disclosure of Potential Conflicts of Interest spreading of metastasis (8, 33, 35-37). Finally, JNJ-26481585 J. Arts: stock owner, Johnson & Johnson. The other authors disclosed showed strong impairment of the function of Hsp90, also a no potential conflicts of interest. substrate of HDAC6. It should be noted though that other – HDAC family members also regulate Hsp90. The class I specif- Acknowledgments ic HDAC inhibitors MS-275 and depsipeptide disrupt Hsp90 function in an indirect manner, through HDAC1-mediated We thank the chemists Sven van Brandt, Marc Willems, and Leo Backx, acetylation and inhibition of Hsp70 function (38, 39). Similar- and Steve McClue for critical reading of the manuscript.

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JNJ-26481585: Novel “Second-Generation” HDAC Inhibitor

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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, et al.

Clin Cancer Res 2009;15:6841-6851. Published OnlineFirst October 27, 2009.

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