Quantitative Analysis of Histone Deacetylase-1 Selective Histone

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Quantitative Analysis of Histone Deacetylase-1 Selective Histone Modifications by Differential Mass Spectrometry Anita Y. H. Lee,† Cloud P. Paweletz,‡ Roy M. Pollock,§ Robert E. Settlage,†,¶ Jonathan C. Cruz,⊥ J. Paul Secrist,§,# Thomas A. Miller,§ Matthew G. Stanton,| Astrid M. Kral,§ Nicole D. S. Ozerova,§ Fanyu Meng,† Nathan A. Yates,† Victoria Richon,§,3 and Ronald C. Hendrickson*,† Department of Proteomics, Merck Research Laboratories, Merck & Co. Inc., Rahway, New Jersey 07065, Department of Proteomics, Merck Research Laboratories, Merck & Co. Inc., Boston, Massachusetts 02115, Department of Cancer Biology and Therapeutics, Merck Research Laboratories, Merck & Co., Inc. Boston, Massachusetts 02115, Boston Drug Design & Optimization, Merck Research Laboratories, Merck & Co., Inc. Boston Massachusetts 02115, and Department of Pharmacology, Merck Research Laboratories, Merck & Co., Inc. Boston, Massachusetts 02115 Received July 8, 2008

Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors. Keywords: HDAC1 • histones • biomarker • mass spectrometry • proteomics • differential mass spectrometry

Introduction Histone deacetylase (HDAC) enzymes catalyze the removal of acetyl groups from the lysine residues on proteins, including histones. There are currently 18 known mammalian histone deacetylase enzymes that can be subdivided into at least three different classes, based on the homology of their catalytic * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Proteomics, Merck & Co. Inc., Rahway, NJ. ‡ Department of Proteomics, Merck & Co. Inc., Boston, MA. § Department of Cancer Biology and Therapeutics, Merck & Co. Inc. ⊥ Department of Pharmacology, Merck & Co. Inc. # Current address: Archemix Corp., Cambridge, Massachusetts, USA. | Boston Drug Design & Optimization, Merck & Co. Inc. 3 Current address: EpiZyme, Inc., Cambridge, MA. ¶ Current address: Virginia BioInformatics Institute, Blacksburg, VA 24060. 10.1021/pr800510p CCC: $40.75

 2008 American Chemical Society

domains to the yeast HDAC enzymes. Class I enzymes which include HDAC 1-3 and 8, are ubiquitously expressed, and predominantly nuclear. Class II enzymes including HDAC 4-7, 9 and 10 show more tissue-specificity, and may exist in both the nucleus and the cytoplasm. Class III HDACs are a structurally distinct class of nicotinamide adenine dinucleotide (NAD+)dependent enzymes. Inhibition of HDAC activity leads to histone hyperacetylation, gene expression changes, and has been shown to induce differentiation, cell cycle arrest, or apoptosis in several cancer cell types.1 As a result, HDACs are promising therapeutic targets for the treatment of cancer, and several HDAC inhibitors targeting ClassI/II enzymes are currently being tested in the clinic. The most advanced of these, vorinostat (suberoylanilide hydroxamic acid [SAHA]), was recently approved for the treatment of Cutaneous T-cell Journal of Proteome Research 2008, 7, 5177–5186 5177 Published on Web 10/31/2008

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Table 1. In vitro Selectivity of the HDAC Inhibitors Used in This Study

a

HDAC IP (nM) compound

MRLB-38489 MRLB-88183 MRLB-07266 MRLB-32353 MS-275b MRLB-63397 HC-Toxin Vorinostat

class

HDAC1

HDAC3

HDAC6

Biaryl Benzamide26 Biaryl Benzamide Biaryl Benzamide25 Benzamide30 Benzamide29 Benzamide25 Cyclic Tetrapeptide27 Hydroxamic acid4

8.4 56.2 19.5 86.4 123.7 89.6 0.8 37.1

9767 10740 >50000 499.3 373.6 438.3 1.1 44.6

>50000 >50000 >50000 >50000 >50000 >50000 4789 40.9

selectivity

HDAC1

HDAC1/3

HDAC1/3/6

a

Assays were performed as described using purified HDAC complexes. Inflection Point (IP) represents the concentration at which 50% of maximal inhibition was achieved. b The studies performed with MS-275 were in support of biomarker studies of a potential clinical candidate.

lymphoma.2,3 Vorinostat targets multiple Class I and Class II HDAC enzymes including HDAC1-3, and 6 (Table 1).4 More recently, there have been efforts to develop compounds targeting specific subclasses of HDACs thought to play a key role in the survival and proliferation of cancer cells. Such compounds should retain anticancer activity, while reducing potential side effects. Several lines of evidence implicate HDAC1 in tumorigenesis; small interfering RNAs (siRNA) targeting HDAC1 have demonstrated a role for this enzyme in the proliferation and survival of tumor cells in vitro5,6 and overexpression of HDAC1 has been demonstrated in gastric, breast, and prostate tumors.7-9 Existing assays that measure HDAC inhibition rely on monitoring histone hyperacetylation; however, histones are substrates for multiple HDAC enzymes, so in order to provide robust cellbased pharmacodynamic readouts of HDAC1 inhibition, it is important to identify those acetyl-lysine residues, or specific patterns of acetyl-lysine residues, that are targeted predominantly, or exclusively by HDAC1. Traditional approaches that are dependent on histonespecific antibody reagents for either histone isolation or immunoblots are not very well suited to analyze global histone post-translational patterns as they are inherently limited by the specificity of the antibody and/or analyze only single posttranslation events. Histones are notoriously and ubiquitously post-translationally modified by phosphorylation; mono-, di-, and trimethylation; ubiquitination; acetylation, and/or sulfonation, and their characterization by mass spectrometry has been the subject of numerous studies. The combination of these post-translational modifications can reach staggering numbers (18 750 potential isoforms for H4 alone).10 Because they contain lysine-rich amino acid sequences which, upon a typical tryptic digestion, result in complex mixtures of amino acid fragments and peptides, each potentially containing a host of post-translational modifications, histone peptides can be difficult to resolve and even more difficult to quantitate. In recent years, several methods have been proposed to circumvent these issues.11 For example, digesting histone proteins with alternative proteases such as Arg-C produced more uniform peptides and allowed for characterization of histone H3 methylation and acetylation states.12 Alternatively, chemical derivatization of lysines and amino groups to propionyl amides with propionic anhydride resulting in trypsin cleavage only after C-terminal arginines followed by conversion of the peptides to d0/d5-ethyl esters successfully quantified differentially expressed histone post-translational modifications in a variety of systems.11 In addition to this and other chemical derivatization strategies, the use of relative ratios of ECD fragment ions from top-down analysis of histone proteins has also been implemented in quantitation strategies.13 The use of mass 5178

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spectrometry coupled with a reproducible histone isolation methodology offers a method to identify the location of posttranslational modifications on a global scale. Although the importance of post-translational characterization of histones by mass spectrometry has recently been demonstrated by both top-down and bottom-up approaches1,14-17 as described above, the identification of a histone-based pharmacodynamic marker, however, requires the identification of compound-specific quantitative changes in the post-translational state of the histone. Most quantitative mass spectrometry requires some type of labeling strategy, either through metabolic incorporation (e.g., stable incorporation of essential amino acids; SILAC18), chemical conjugation (e.g., isotope coded affinity tags, ICAT, iTRAQ19), or deuterium label incorporation during enzymatic digestion or esterification of glutamic and aspartic acids.20 Some of these protein manipulations are 30 years old, and therefore have been proven to be quite dependable and reliable. However, these approaches are not easily adapted for comparisons between many treatment groups or multifactorial comparisons. To help circumvent these issues, we have developed a general workflow called differential mass spectrometry (dMS).21-23 Differential mass spectrometry (dMS) describes a new class of experiments that combine reproducible sample processing, high resolution mass spectrometry, and statistical analysis into an efficient and general approach for the quantification and identification of biologically relevant analytes in complex mixtures. This approach is not limited by MS/MS data acquisition rates and supports complex experimental designs. Here, we describe the use of dMS to identify features representing histone post-translational modifications that change in response to HDAC- inhibitors of varying degrees of selectivity, including a subset that are highly specific for HDAC1. HCT116 human colon cancer cells were treated with inhibitors of HDAC1, HDAC1/3, or HDAC1/3/6 in 2 independent experiments that included several different chemical classes (Table 1). With the use of dMS, we found 24 features that changed in response to treatment with HDAC1-selective inhibitors. These ions were targeted in a separate MS/MS run, and using high resolution linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTICR), we were able to deduce the amino acid sequence and acetylation site for 7 peptides. The methods described in this manuscript demonstrate a generally applicable procedure, and illustrate the promise of dMS for biomarker discovery, specifically the identification of post-translational patterns that may not be immediately ad-

HDAC1 Selective Peptides by Proteomics

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Figure 1. (A) Differential mass spectrometry (dMS) workflow. Histones isolated from individual biological replicates are digested to their tryptic peptides, profiled by LC-FTMS mass spectrometry, and statistically significant ion intensities that differ between samples from distinct treatment conditions are determined by dMS as described in Materials and Methods. Those ions are subsequently targeted in separate run for MS/MS identification. (B) Receiver-operator curve (ROC). ROC curves for Vehicle A vs vorinostat and Vehicle B vs vorinostat comparisons are shown. The false positive results for these comparisons were few and far less statistically significant indicating reproducibility of the overall platform.

dressable by antibody reagents. We envision that LC-MS based protein profiling will lead to the identification of novel peptide biomarkers.

Materials and Methods HDAC Biochemical Activity Assay. Carboxy-terminal FLAGtagged human HDACs 1, 3 (coexpressed with a His-tagged SMRT (aa 1-899)), and 6 were overexpressed in mammalian

cells and affinity purified using an anti-Flag antibody matrix, eluted from the matrix with 100 µg/mL of a competing FLAG peptide in 20 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10% glycerol, and protease inhibitor cocktail (Roche cat. # 1836153). As the HDAC3 complex may contain HDAC1, it was further purified over an affinity column directed against HDAC1 to remove these complexes. The HDAC Fluorescent HDAC1 Fluor-de-Lys Activity Assays from BioMol Research Laboratories (Plymouth Journal of Proteome Research • Vol. 7, No. 12, 2008 5179

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Figure 2. Example of an acetylated histone peptide identified in HCT116 cells treated with vorinostat. The dMS output graphs (A-C) and MS/MS spectra (D) for peptide KAcQLATKAcAARAc (m/ztheoretical ) 535.8195; m/zobserved ) 535.8175; Mass accuracy ) 3.73 ppm) from histone H3 acetylated at lysine 18 and 23 are shown. Panel A shows the chromatographic profile in where the solid line is the mean intensity between groups, the dotted lines the standard error of the mean, and the vertical dotted lines show the region of significance. Panel B shows a bar graph consisting of the intensity along with standard error for the same feature, while panel C shows the individual ion chromatogram for each replicate. (*** p < 0.001.) Table 2. Compounds Used for the First HDAC1-Selective dMS Profiling Experiment compound

concentration

class

selectivity

Vehicle MRLB-38489 MRLB-32353 Vorinostat

10µM 10µM 10µM

Biaryl Benzamide Benzamide Hydroxamic acid

Control HDAC1 HDAC1/3 HDAC1/3/6

Meeting, PA) provided the basis for our HDAC activity assays. The assay buffer for the HDAC1 assay consisted of 20 mM Hepes, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1 mg/mL BSA; for the HDAC3 assay, it was 20 mM Hepes, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.25 mg/ mL BSA, and for the HDAC6 assay, it was 20 mM Hepes, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1 mg/mL BSA. A total of 3× serial dilutions of 10 mM compound were performed in DMSO followed by a 20× dilution into Assay Buffer. Twenty microliters of HDAC was preincubated with 5 µL of diluted compound at room temperature for 10 min. The reaction was initiated by the addition of 25 µL of the substrate (BIOMOL catalog # KI-104), incubated 60 min at 37 °C, before adding 50 µL of the development solution (167×-diluted 20× Developer Concentrate (BIOMOL: KI-105) plus 10 µM SAHA). 5180

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The assay was read in a VictorV plate reader (Perkin-Elmer, Wellesley, MA) at Ex 360 nm/Em 460 nm. Cell Culture and Histone Extraction. To determine the overall reproducibility and false positive rate of dMS analysis on histones isolated from cell culture, 10 individual experimental replicates of HCT116 human colon cancer cells were treated with Vehicle (0.01% DMSO) and five experimental replicates were treated with vorinostat (10 µM) for 4 h. For subsequent experiments, cells were treated in triplicate with HDAC-specific compounds at the concentrations listed in Tables 2 and 4 for 12 and 24 h, respectively. Compound concentrations and incubation times were designed to maximize differential HDAC inhibition, minimize compound toxicity during the experiment, and allow matched pair compounds to be used at equivalent concentrations. Approximately 2.5 × 106 to 107 cells were harvested and washed once with 1 mL of cold PBS. The cells were pelleted by centrifugation at 600g for 5 min. The pellet was then resuspended in 1 mL/107 cells of ice cold Lysis Buffer (10 mM MgCl2, 10 mM Tris-HCl, 25 mM KCl, 1% TritonX-100, 8.6% sucrose, two tablets of protease inhibitors (Roche)/100 mL, pH to 6.5) and incubated on ice for 5 min. After incubation, the cells were centrifuged at 600g for 5 min at 4 °C, the supernatant was removed, and the pellet was resuspended in 1 mL/107 cells of TE Buffer (10 mM Tris-

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Table 3. Number of HDAC1 and HDAC3-Selective Features Found in the First dMS Experiment

a

(***) p < 0.001.

Table 4. Compounds Used for the Second HDAC1-Selective dMS Profiling Experiment compound

concentration

class

Vehicle MRLB-88183 MRLB-07266 MS-275 MRLB-63397 HC-Toxin Vorinostat

1 µM 5 µM 1 µM 5 µM 50µM 2 µM

Biaryl Benzamide Biaryl Benzamide Benzamide Benzamide Cyclic Tetrapeptide Hydroxamic acid

selectivity

Control HDAC1 HDAC1/3

HDAC1/3/6

HCl, pH 7.4, 0.5 M EDTA, pH 7.4). The cells were then centrifuged again at 600g for 5 min at 4 °C, the supernatant was removed, and the pellet was resuspended in 100 µL of icecold water containing 0.4 N H2SO4 and vortexed for 5 s. The lysate was incubated on ice for 1 h and vortexed for 10 s every 15 min during the incubation. The sample was then centrifuged at 10 000g for 10 min at 4 °C and the supernatant was collected. Ice-cold acetone was added to the collected supernatant at 1 mL/100 µL of supernatant and incubated at -20 °C for g1 h. Following incubation, the sample was centrifuged at 10 000g for 10 min at 4 °C and the supernatant was carefully removed without disturbing the translucent pellet. The tube was inverted on an absorbent pad and allowed to air-dry. The precipitated histone pellet was resuspended in water (∼100 µL/107 cells) and the protein concentration was measured by a Bradford assay (Bio-Rad, Hercules, CA). Trypsin Digestion. For each treatment condition, 5 µg of total histone protein was reduced in 4 mM TCEP (Pierce, Rockford, IL) and digested overnight at 37 °C with 125 ng of Trypsin Gold (Promega, Madison, WI). All solutions were made in 50 mM ammonium bicarbonate, pH 8. Digested peptides

were dried in a Speed-Vac and resuspended in 0.1 M acetic acid prior to LC-MS analysis. Liquid Chromatography and Electrospray Ionization Hybrid Linear Ion Trap-FTMS. LC-MS analysis was performed as previously described.21 Briefly, samples were analyzed by LC-MS using a HP1100 micro capillary pump (Agilent, Palo Alto, CA), a FAMOS autosampler (Dionex, Sunnyvale, CA) and a linear ion trap-FTMS (Thermo Electron, San Jose, CA). Over the peptidic region, one FTMS full scan, 3 data-dependent ion trap MS/MS, and 1 ion trap full scan were acquired repeatedly (the total cycle time was ∼0.9-1 s). Typical instrument settings were spray voltage ) 3.0 kV; AGC settings ) 1 × 106 (for FTMS full scan); maximum injection time ) 1.0 s (for FTMS full scan); and resolution ) 50 000. To account for any systematic chromatographic and mass spectrometric performance changes over time, samples from different treatment conditions were interleaved. Analysis of LC-FTMS Data by dMS. Differential mass spectrometry is a general proteomics workflow that provides relative quantitation and identifies statistically significant changes in full scan mass spectrometry data.21-23 Briefly, the LC-MS data is aligned, normalized and gridded to generate an intensity measurement at each m/z and retention time. In these experiments, the nonparametric Kruskal test is used to determine statistically significant data points. Signals that arise from different charge states and isotopes of a single analyte are grouped together to generate a ranked list of m/z values that are statistically different across the two groups. To find the false positives, mass spectrometric data from the first five Vehicle samples (A) were compared with the second five Vehicle samples (B) by dMS. Data from the Vehicle treated samples were compared with data from the vorinostat treated samples to find features that arise from vorinostat treatment. Data from subsequent experiments were either compared as Journal of Proteome Research • Vol. 7, No. 12, 2008 5181

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Table 5. Number of HDAC1 and HDAC3-Selective Features Found in the Second dMS Experiment

a

(**) p < 0.01; (***) p < 0.001.

Figure 3. Defining HDAC1-specific peptides by profiling multiple structurally diverse compounds. Shown is the number of overlapping features identified to be HDAC1-specific in 2 independent dMS profiling experiments. A total of 3 distinct HDAC 1 compounds were compared against 4 HDAC 1/3-specific and 1 HDAC 1/3/6-specific inhibitor (see Table 1).

individual treatment groups by dMS or combined based on their in vitro HDAC-specificity and then compared by dMS. Peptide Identification by Database Searching. Features of interest were selected for targeted tandem MS/MS analysis in a subsequent run in order to identify the peptide sequence. Collected MS/MS spectra were searched using SEQUEST against a human histone database downloaded from the NCBI with differential mass modifications of +42 Da (acetylation, trimethylation) and +14 Da (monomethylation) or +28 (dimethylation) on lysine residues, and +80 Da (phosphorylation) on serine, threonine, and tyrosine residues. Peptide sequences were validated manually and the mass accuracy of the parent ion was confirmed. In Vitro Validation by Western Blotting. HCT116 cells were treated for 24 h with or without 1 µM MRLB-38489. Histones were extracted as described above and separated (4 µg/lane) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 18% Criterion Pre-Cast polyacrylamide gels (BioRad, Hercules, CA). Gels were transferred onto 0.45-µm nitrocellulose membranes (Invitrogen, Carlsbad, CA). Rabbit polyclonal anti-acetyl-lysine antibodies specific for histone residues 5182

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H2B-K5 (Catalog No. 2574), H3-K18 (Catalog No. 9675), H3K23 (Catalog No. 9674), and H4-K8 (Catalog No. 2594) were purchased from Cell Signaling Technologies (Beverley, MA), apart from the antibody specific for acetylated-H4-K5 (Catalog No. 06-759), which was purchased from Upstate Cell Signaling Solutions (Temecula, CA). All primary antibodies were used at a 1:1000 dilution. Goat anti-rabbit secondary antibody conjugated to Cy5 fluorescent label (Catalog No. 81-6116) was purchased from Zymed Laboratories Inc. (San Francisco, CA) and used at a 1:7000 dilution. Blots were imaged on an Amersham Typhoon 9410 Imager using the Cy5 laser settings. Histones were also analyzed by GelCode Blue stain (Pierce, Rockford, IL). In Vivo Validation in HCT116 Xenograft-Bearing Mice. Exponentially growing HCT-116 were trypsinized as previously described and resuspended in equal volumes of ice-cold PBS and Matrigel (Becton, Dickson and Company, Franklin Lakes, NJ). Cells were then counted in a Vi-Cell XR cell viability analyzer (Beckman Coulter, Fullerton, CA), and 5 × 106 cells/ 100 µL were injected subcutaneously (sc) into the right flank of 4-6 week old female CD-1 nude mice (Charles River Laboratories, Wilmington, MA). The mice were housed under laminar flow with water and food available ad libidum. All mice were examined daily and sc tumor volumes were measured twice weekly. Between 90-95% of sc implanted mice developed palpable tumors within 2 weeks of sc injection. When tumors reached a volume of 300-400 mm3, a single intraperitoneal injection (10 mL/kg) of vehicle (10% DMSO/45% PEG400/45 %H20) or compound (MRLB-38489, 100 mg/kg) was given to the sc xenograft-bearing mice (n ) 4-5 per group). At predetermined time points, sc tumors were harvested and frozen in liquid nitrogen. Histones were extracted from the tumors and acetylation levels were determined by indirect ELISA using various antibodies described in the previous

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Table 6. Amino Acid Sequence for the 7 of 24 HDAC1-Selective Features Identified

a

(**) p < 0.01; (***) p < 0.001, K# is an acetylated lysine residue.

section. Reported results are representative of experiments repeated at least three times.

Results and Discussion Platform Evaluation Using Receiver-Operator Curves (ROC). The general dMS workflow is shown in Figure 1A. Histones isolated from individual biological replicates are digested to their tryptic peptides, profiled by LC-FTMS mass spectrometry, and ion intensities that show statistically significant differences between the treatment conditions are determined by dMS. As the analysis is performed on the full scan MS spectra, no derivatization of histone residues is necessary. Instead, we exploited the fact that trypsin does not cleave after acetylated lysine residues;24 thus, the resulting tryptic peptides of histones from cells treated with vehicle will be distinctly different than those treated with a histone deacetylase inhibitor. To test the overall reproducibility, and false positive rate of our platform for histone profiling, we designed an experiment where we performed dMS analysis on Vehicle versus Vehicle and Vehicle versus vorinostat-treated histone fractions with 5 individual experimental replicates, and

generated receiver-operator characteristic (ROC) curves. Comparison of the samples from the vorinostat-treated (>250 times in vitro IP for HDAC1, see Table 1) cells versus one set of the vehicle samples was used to estimate sensitivity, while the comparison between two groups of vehicle samples was used to estimate specificity. The ROC curves display the tradeoff between sensitivity and specificity in the following way: as the p-value threshold is sequentially raised, the ROC curve shows how the increase in sensitivity (more differences detected between treatment and vehicle) is offset by the decrease in specificity (as measured by the increase in vehicle vs vehicle comparisons called different). The ROC curve is shown in Figure 1B. As can be seen, the false positive results for these comparisons were few and far less statistically significant indicating that our biochemical and mass spectrometry platform is highly reproducible. The analysis was repeated for a second set of vorinostat versus Vehicle treated cells and showed essentially the same results (Figure 1B). On the basis of these results, we conclude that the entire experimental design (isolation of histones to mass spectrometric profiling) is highly reproducible and is capable to broadly Journal of Proteome Research • Vol. 7, No. 12, 2008 5183

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Figure 4. Identification of PEPAKAcSAPAPK as a peptide modulated by HDAC1-specific inhibition. The dMS output graphs (A-C) and MS/MS spectra (D) of PEPAKAcSAPAPK m/z 567.8 (m/ztheoretical ) 567.8113; m/zobserved ) 567.8110; Mass accuracy ) 0.53 ppm) from histone H2B.a acetylated at lysine residue 5 is shown. Panel A shows the averaged chromatographic profile; panel B, the graphical output from dMS as intensity; panel C, the individual ion chromatograms. (*** p < 0.001.)

Figure 5. Western blot analysis of specific histone lysine residue hyperacetylation after HDAC1-inhibition of HCT116 cells. Histones were isolated from HCT116 cells treated for 24 h with, or without, 1 µM MRLB-38489 and immunoblotted with antibodies to the indicated acetyl-lysine residues.

Figure 6. Confirmation of Ac-H2B (K5) as a readout for HDAC1selective inhibition in HCT116 human cell line xenografted mice. Shown are ELISA results monitoring the acetylation status of specific histone lysine residues (Ac-H3-K9/14, Ac-H4-K8, and AcH2B-K5) in tumors isolated from mice 7 h after MRLB-38489 dosing. The average drug concentration in tumor and plasma was determined to be 15.1 and 14.5 µM, respectively. The calculated fold-change in acetylation levels relative to vehicletreated mice was 3.4, 4.8, and 11.2 for Histone H3, H4, and H2B, respectively. Statistical significance was determined by an unpaired, two-tailed t test (* p < 0.05; ** p < 0.01).

profile the post-translational status of histones in this experimental system.

Next, we set out to identify a subset of features that were different between the vehicle-treated as compared to the vorinostat-treated group. Data-dependent tandem MS/MS

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HDAC1 Selective Peptides by Proteomics spectra were submitted to SEQUEST for searching against a human histone database generated from the NCBI database. Most of the peptides observed were histones and showed statistically significant differences in their acetylation status. Figure 2 shows an example of a ‘feature’ that changes with vorinostat treatment. The amino acid sequence derived from the high mass accuracy MS/MS spectra reveals histone H3 is acetylated at lysine residues 18 and 23. The theoretical mass of the (M + 2H)+ ions ) 535.8195 agrees with the observed mass of the (M + 2H)+ ions ) 535.8175 (mass accuracy ) 3.73 ppm). Since trimethylated peptides and acetylated peptides differ in mass by 34 ppm, we conclude that this peptide is acetylated rather than trimethylated. Identification of Features Corresponding to HDAC1-Selective Inhibition by Differential Mass Spectrometry. Having characterized the platform to profile histones, we then initiated a dMS profiling experiment to find features that changed with HDAC1-selective inhibitors. Three compounds that have been shown to inhibit different HDAC subfamilies in vitro were selected (Table 2). We used the HDAC1-selective compound MRLB-38489 at a concentration of 10 µM based on an initial measurement of an HDAC3 in vitro IP of >50 000 nM. However, in assays performed after completion of these experiments, the HDAC3 in vitro IP is consistently found to be closer to 10 000 nM (Table 1). Nevertheless, the differential effect of MRLB38489 on HDAC1 relative to HDAC3 should still be large in this experiment. Following dMS analysis, we categorized HDAC1selective features as those that are shared by all compounds, and HDAC3-selective features as those that are shared by vorinostat and MRLB-32353, but not by MRLB-38489. Using these criteria, we found 135 putative HDAC1-selective features and 29 putative HDAC3-selective features (Table 3). To gain confidence that these peptide features were generally applicable to HDAC1-selective inhibitors, we extended the number of compounds from the first dMS profiling experiment. The second dMS experiment contained vorinostat, as well as 2 and 3 additional compounds that were shown to be HDAC1and HDAC 1/3-specific, respectively. Compounds and corresponding concentrations are shown in Table 4. The experiment was designed to include compounds with similar core structures, but with different degrees of HDAC-selectivity. For example, MRLB-07266 (HDAC1-selective) and MRLB-63397 (HDAC1/3-selective) both possess a benzothiophene core,25 and MRLB-38489 (HDAC1-selective) and MS-275 (HDAC1/ 3-selective) both contain the same core structure, apart from the specificity-determining R-groups.26 For dMS analysis, we grouped compounds based on selectivity (i.e., HDAC1, HDAC1/ 3, HDAC1/3/6). Following dMS analysis, we categorized HDAC1selective features as those features that are shared by all compounds, and HDAC3-selective features as those that are shared by vorinostat and MS-275, MRLB-63397, and HCToxin,27 but not by MRLB-88183 or MRLB-07266. Using these criteria, we found 31 HDAC1-selective features and 14 HDAC3selective features (Table 5). To investigate which HDAC1specific features were shared between both experiments, we determined which features overlapped between the two independent experiments. As shown in Figure 3, 24 out of 31 peptides were repeatedly found to be differentially regulated by HDAC1-specific compounds. Seven of these 24 HDAC1selective features were sequenced. These include acetylated peptides from histones H4/O, H2B.B, H2B.N, H2B.E, and H3 as shown in Table 6. Figure 4 shows one of the HDAC1-selective features identified as a histone H2B.a peptide acetylated on

lysine residue 5 with a mass accuracy of 0.53 ppm. The number peptides sequenced, 7/24, in this study is small but not completely unexpected. The overall success rate depends on the ability to isolate the targeted precursor ion cleanly, and the completeness of the database, both of which can be limiting factors. In Vitro and in Vivo Validation of Readouts for HDAC1Selective Inhibitors Using Specific Histone Acetyl-lysine Antibodies. We then set out to confirm whether HDAC1-specific peptides that were identified in this dMS experiment could be used as readouts of HDAC1-selective inhibition. To this end, we used commercially available antibodies to monitor the acetylation status of specific lysine residues contained within the HDAC1-specific peptides in cells treated with an HDAC1selective inhibitor. Histones were isolated from HCT116 cells treated in culture for 24 h with, or without 1 µM MRLB-38489. Changes in the acetylation status of H2B-K5, H3-K18, H3-K23, H4-K5, and H4-K8 in response to MRLB-38489 treatment were monitored by immunoblotting with acetyl-lysine specific antibodies. The results, shown in Figure 5, show that acetylation at each histone lysine residue showed a clear increase in response to HDAC1-selective inhibition similar to the dMS analysis results. To investigate the utility of a subset of these acetyl-lysine residues as pharmacodynamic markers of HDAC1 target engagement in vivo, histones were extracted from HCT116 xenografted tumors in mice 7 h after dosing with MRLB-38489, or vehicle control. ELISA assays on the extracted histones using antibodies to Ac-H3-K9/14, Ac-H4-K8, and Ac-H2B-K5 are shown in Figure 6. Compellingly, both Ac-H4-K8 and Ac-H2BK5 antibodies gave stronger signal in this assay than another antibody commonly used to track HDAC inhibitor activity. This antibody recognizes a H3 peptide acetylated at residues K9 and K14 that was not identified in our HDAC1-selective dMS profiling experiments. These results provide in vivo validation of the profiling results, and confirm that Ac-H2B-K5 represents a robust pharmacodynamic marker for HDAC1-inhibition in this xenograft model.

Conclusions The use of mass spectrometry coupled with reproducible sample preparation offers a promising method to identify large numbers of post-translational changes of histones. Here, we demonstrate the use of this general workflow, differential mass spectrometry (dMS), to profile histones isolated from the HCT116 human colon cancer cell line that have been treated with either a multi-HDAC inhibitor vorinostat or 7 HDACselective compounds incorporating several distinct chemical classes. We note that the 7 compounds used in this experiment may well cover only a very small part of the “chemical space” of HDAC 1 specific acetylation patterns, so we do not know whether these patterns would generalize to a larger number of compounds; nonetheless, one of the peptides found showed a clear pharmacological response in a relevant xenograft model. Our findings are furthermore supported by observations from a recent paper that shows that siRNA silencing of HDAC1 greatly increases acetylation of H3-K18 in HeLa cells, whereas knockdown of HDAC2 or HDAC3 had a much smaller effect28 implicating histone H3 acetylation at lysine residue 18 regulation by HDAC1-specific compounds.

Acknowledgment. Special thanks to Matthew Wiener for generating the ROC curves, the anonymous reviewers for Journal of Proteome Research • Vol. 7, No. 12, 2008 5185

research articles helpful comments on the manuscript, and Steven Friend, Alan Sachs, and Kevin Chapman for executive sponsorship and support.

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