Label-Free in Situ Monitoring of Histone Deacetylase Drug Target

Feb 21, 2014 - Here, we demonstrate that label-free quantification of histone ... The large number of compounds that are currently being tested in the...
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Label-Free in Situ Monitoring of Histone Deacetylase Drug Target Engagement by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Biotyping and Imaging Bogdan Munteanu,†,‡,§ Björn Meyer,†,‡,§ Carolina von Reitzenstein,†,‡,§ Elke Burgermeister,∥ Susanne Bog,⊥ Andreas Pahl,⊥ Matthias P. Ebert,∥ and Carsten Hopf†,‡,§,* †

Institute of Medical Technology, University of Heidelberg and Mannheim University of Applied Sciences, ‡Center for Applied Research in Biomedical Mass Spectrometry (ABIMAS), and §Instrumental Analytics and Bioanalytics, Mannheim University of Applied Sciences, Paul-Wittsack-Strasse 10, 68163, Mannheim, Germany ∥ Department of Medicine II, Medical Faculty Mannheim, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany ⊥ Heidelberg Pharma GmbH, Schriesheimer Strasse 101, 68526 Ladenburg, Germany S Supporting Information *

ABSTRACT: Measurements of target activation in cells or tissues are key indicators of efficacy during drug development. In contrast to established methods that require reagents and multiple preprocessing steps, reagent-free in situ analysis of engaged drug targets or target-proximal pharmacodynamic signatures in solid tumors remains challenging. Here, we demonstrate that label-free quantification of histone acetylation-specific mass shifts by matrix-assisted laser desorption ionization (MALDI) mass spectrometry biotyping can be used for measurement of cellular potency of histone deacetylase inhibitors in intact cells. Furthermore, we employ MALDI mass spectrometry imaging of these mass shifts to visualize the spatiotemporal distribution of acetylated histones and thus the tumor-selective pharmacodynamic responses in a mouse model of gasterointestinal cancer. Taken together, our study suggests that the monitoring of drug-induced mass shifts in protein ion intensity fingerprints or images may be a powerful analytical tool in pharmacology and drug discovery.


Protein families, which add, remove, or recognize posttranslational modifications (PTM) on DNA or histones and thereby modulate gene transcription through epigenetic mechanisms, have arguably become one of the promising frontiers for drug discovery.10 Histone deacetylase inhibitors (HDACi) that block the deacetylation of defined acetyllysine moieties in histone Nterminal tails are the most advanced class of epigenetic drugs, as suberanilohydroxamic acid (SAHA; vorinostat) and romidepsin have been approved for treatment of cutaneous T-cell lymphoma. The large number of compounds that are currently being tested in the clinic, including the hydroxamic acid LBH589 (panobinostat) and the aminobenzamide CI-994 (tacedinaline), display remarkable differences in isoform and target protein complex selectivity suggesting possible differences in clinical pharmacology.7,11−13 We therefore chose HDACi as an example for validation of a new method for label-free in situ monitoring of drug target engagement by MALDI-TOF MS. To this end, we developed a direct, quantitative profiling technique for HDACi-induced histone PTM in cancer cells

rug discovery and development remain challenging, expensive, and time-consuming endeavors, and a substantial number of drug candidates with acceptable physicochemical, pharmacokinetic, and toxicological properties fail in preclinical or clinical efficacy studies.1,2 It has been argued that more direct measurements of drug target occupancy and activation, collectively referred to as target engagement, should form the basis of mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) modeling and thereby of fundamentally improved drug efficacy studies.2−5 In situ monitoring of drug activity could thereby facilitate treatment selection, monitoring, and response prediction in personalized strategies. Array- and mass spectrometry (MS)-based workflows for identification and measurement of target activation/engagement markers have been extensively employed in recent years for various drug target classes.3,6,7 Most recently, activity-based proteomic probes and probe-independent cellular thermal shift assays have been introduced as methods for monitoring drug target occupancy/engagement.8,9 However, all currently available methods require cell/tissue extraction, multistep sample processing, and frequently expensive reagents. © 2014 American Chemical Society

Received: January 3, 2014 Accepted: February 21, 2014 Published: February 21, 2014 4642 | Anal. Chem. 2014, 86, 4642−4647

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Figure 1. Label- and reagent-free quantitative profiling of epigenetic drugs by whole cell MALDI MS Biotyping in K562 cells. (a,b) Time-dependent shifts (Δm/z = 42) in averaged WC-MALDI MS fingerprints at m/z 11 306 indicate progressive histone H4 poly acetylation (0 Ac to 4 Ac) after SAHA treatment (20 μM). Note that the half-life (∼2 h) of the nonacetylated state can be determined. (c) Concentration-dependence of histone H4 hyperacetylation in response to SAHA (0.3 and 30 μM). (d) Gel-view illustrates a concentration-dependent increase in histone H4 acetylation after treatment with SAHA for 24 h. (e) Concentration−response curves derived from WC-MALDI MS fingerprint data enable determination of EC50 values for different HDACi. Ion intensities of the peak corresponding to nonacetylated histone H4 (normalized to vehicle control) from three independent measurements were subjected to a nonlinear regression curve fit.

acetyl moeity differs from a trimethyl moeity by only 0.03639 Da. However, SAHA and CI-994 are well-characterized inhibitors known to not modulate demethylases. Furthermore, the major methylation site on H4, K20, is basally dimethylated,18 and the m/z we refer to as polyacetylation states are consistent with dimethylated H4. We therefore concluded that mass shifts of 42 Da corresponded to lysine acetylation. H4 acetylation progressed from a predominantly nonacetylated (H4 0 Ac) to a predominantly 4-fold acetylated (H4 4 Ac) state. More importantly for applications in drug discovery such as compound screening or profiling, m/z shifts for H4 and ion intensities of peaks corresponding to its various acetylated states were reproducible, concentration-dependent and enabled the determination of EC50s of 10 nM, 0.54 μM, and 1.23 μM for the HDACi LBH-589, SAHA, and CI-994, respectively (Figure 1c−e and Supplementary Figures S6 and S7 in the Supporting Information). Moreover, determination of EC50s by quantification of the H4 0 Ac peak was not limited to suspension cells, but it could also be measured for the adherent breast cancer cell line MDA-MB-468 that required trypsinization during harvesting (Supplementary Figure S8 in the Supporting Information). EC50s for LBH-589 were in line with values (10−50 nM) obtained with recombinant enzymes or in other lymphoma cell lines using assays of cell survival and of distal HDACi effects, e.g., PARP cleavage.12,17 Encouraged by these results, we sought to translate this approach to in vivo studies in cancer animal models. Even the most recent methods for monitoring of in vivo drug target engagement8,9 do so by analyzing extracts that do not preserve spatial information of the tissue. Pote et al. have recently combined protein patterns obtained by MALDI-IMS with LC−

utilizing whole cell (WC)-MALDI-MS Biotyping and successfully transferred it to MALDI-imaging MS (IMS) of HDACi action in solid tumors. We reasoned that WC-MALDI MS Biotyping, i.e., the direct and reagent-free analysis of mammalian cells homogenized in a solvent/MALDI matrix mixture, could be adapted for monitoring of histone PTM and that it would be robust enough for label-free quantitation and determination of compound EC50s in cell-based assays14,15 (Supplementary Figure S1 in the Supporting Information). However, m/z values corresponding to histones (as determined by liquid chromatography−tandem mass spectrometry (LC−MS/MS) in an earlier study16) were barely detected in MS fingerprints obtained by published biotyping methods.14,16 Optimization of solvent acidity and composition affected extraction and cocrystallization of all four core histones and consequently improved signal intensity and technical reproducibility of histone measurement (Supplementary Figure S2 and Supplementary Table S1 in the Supporting Information). We incubated K562 chronic myelogenous leukemia suspension cells, which we have recently used to study HDACi action,7 with SAHA for up to 24 h and recorded MS fingerprints. Whereas their overall appearance was unaffected by SAHA treatment, peaks corresponding to histones H2A, H2B, H3, and H416 were markedly shifted (Figure 1a and Supplementary Figures S3 and S4 in the Supporting Information). Multiple peaks corresponding to dimethylated histone H4 (m/z 11306; others with Δm/z = 42) that indicated time-dependent polyacetylation of this histone in response to SAHA and CI994 were MS-resolved (Figure 1a,b and Supplementary Figures S3, S4c, and S5 in the Supporting Information). The mass of an 4643 | Anal. Chem. 2014, 86, 4642−4647

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Figure 2. Label-free in situ monitoring of target engagement and of pharmacodynamic effects of LBH-589 in a mouse model of gastric cancer by MALDI imaging MS. (a) LBH-589 administration (30 mg/kg) results in a time-dependent decrease of nonacetylated (0 Ac) histone H4 and a concomitant increase in acetylated H4 states (H4 2 Ac to −4 Ac). Note that in vehicle-treated animals tumor tissue is hypoacetylated (H4 0 Ac) when compared to nontumor tissue. (b) Histone H4 hypoacetylation in tissue sections from vehicle-treated mice and H4 hyperacetylation in LBH589-treated mice is predominantly located to tumor tissue, as assessed by H&E staining. Scale bar = 0.25 mm. (c) Single MALDI-IMS spectra of intense pixel (red color in part a from zoom-in regions (in part b) confirm histone hyperacetylation in tumor regions after LBH-589 administration.

acetylation and measurement of drug pharmacodynamics in situ was possible. Toward this end, we treated human MDA-

MS/MS information on histone acetylated states for diagnostic purposes.18 We wondered if direct analysis of histone 4644 | Anal. Chem. 2014, 86, 4642−4647

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purchased from Bruker Daltonics (Bremen, Germany). Vorinostat (SAHA) (catalog no. V-8477) and tacedinaline (CI-994; catalog no. C-2606) were purchased from LcLabs (Woburn, USA); panobinostat (LBH-589) (catalog no. S1030) was obtained from Selleckchem. Frozen Section Compound (FSC 22) was purchased from Surgipath (Richmond, USA). In Vitro Histone Deacetylase Inhibitor Studies by Whole Cell-MALDI TOF MS Biotyping. The chronic myelogenous leukemia cell line K562 was maintained in RPMI-1640 medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, 1.25 g of glucose, 10% fetal calf serum (FCS) (v/v), and 1× penicillin/ streptomycin. The adherent breast cancer cell line MDA-MB468 (Cell Line Systems catalog no. 330279) was maintained in DMEM:Ham’s-F12 (1:1) medium supplemented with 2 mM Lglutamine,10% FCS (v/v), and 1× penicillin/streptomycin. Histone deacetylase inhibitors (HDACi) were dissolved in DMSO at a concentration of 100 mM and stored at −20 °C as stock solutions. To study the effect HDACi on cellular histone acetylation, cells were seeded in 6-well or 24-well plates (CELLSTAR Greiner Bio-One catalog no. 657 185 and 662 102) at a cell density of 500 000 cells/mL 12 h prior to stimulation. Adherent cells were seeded at a cell density of 500 000 cells per well in 6-well plates (CELLSTAR Greiner BioOne catalog no. 657 160) and were allowed to attach overnight. Cells were treated with compounds or vehicle control, diluted 1:1000 in cell culture medium, for concentration-dependence and time-course studies. Adherent cells were detached by trypsinization and collected by centrifugation for 5 min at 2 000 rpm at 4 °C, without additional washing steps, and cell aliquots were snap-frozen in liquid nitrogen and stored at −80 °C until use. Sample preparation for WC-MALDI MS biotyping was performed as described elsewhere14 with slight modifications: Frozen cells were resuspended in ddH2O to obtain cell concentrations of 5 000 cells/μL. A volume of 15 μL of the suspension was mixed with 15 μL of SA (38 mg/mL in water/ ACN/TFA 20/80/2.5 (v/v/v)). Thereafter, 1 μL of the matrix−analyte mix corresponding to 2 500 cells was applied on top of the thin SA layer using the dried droplet preparation (double layer) and air-dried. All mass spectra were acquired on an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a 200 Hz Smartbeam laser using the AutoXecute function of the FlexControl 3.4 acquisition software. Briefly, each spectrum was acquired in positive linear mode within the mass range of m/z 4 000−20 000 with a low mass gate at m/z 3 000. A total of 4 000 laser shots were accumulated for each sample from 20 different positions of the MALDI target chosen by random walk. Acceleration voltage was set to 20 kV and delayed extraction parameters balanced for optimal resolution and sensitivity in the selected mass range. Additional peak quality parameters were full width at halfmaximum (fwhm) < 20 ± 2 Da, minimum S/N > 3, and omission of the two highest peaks. Protein spectra underwent “real time smooth: high” processing in FlexControl 3.4. Subsequently, protein mass spectra were baseline-subtracted and externally calibrated in Flex Analysis 3.4 (Bruker Daltonics) using the protein calibration standard I (Bruker Daltonics). Average MALDI-MS cell fingerprints were generated using ClinProTools 3.4 software (Bruker Daltonics) (CPT) using processing parameters described in details elsewhere.14 In Vivo Pharmacology and Tissue Preparation for Imaging Mass Spectrometry (IMS). Animal studies were

MB-468 xenograft-bearing mice with LBH-589 (30 mg/kg i.p.) or vehicle for 4 h and analyzed cryosections by MALDI-IMS. Following LBH-589 treatment, the ratio of H4 0 Ac to 1 Ac signals was notably inverted, and H4 2 Ac and 3 Ac signals were markedly increased in tissue that histopathology identified as tumorous (Supplementary Figure S9 in the Supporting Information). We have recently elucidated the mechanism by which LBH-589 sensitizes gastric cancer cells to conventional chemotherapeutics in CEA/TAG transgenic mice that develop spontaneous gastric tumors.19,20 To corroborate our results in xenograft tissue and to gain further insight into the in situ action of LBH-589 in this model of gastric cancer, we performed a similar study in these mice. In control mice we observed striking hypoacetylation of H4 in tumor tissue compared to stromal tissue outside the tumor (Supplementary Figure S10 in the Supporting Information). Since H4 hypoacetylation has previously been described as a marker for cancerous tissue,21 this finding also serves as method validation. In contrast, LBH589 treatment caused a time-dependent decrease in nonacetylated H4 and concomitant increase in multiply acetylated H4 stages (Figure 2 and Supplementary Figure S11 in the Supporting Information). Surprisingly, the hyperacetylationinducing activity of LBH-589 was much more pronounced in tumor tissue and minimal in nonmalignant tissue, thus revealing a striking tumor-selectivity of this HDACi. In summary, we provide proof-of-concept for the in situ analysis of pharmacodynamic drug responses with spatial resolution by MALDI-IMS. Furthermore, we demonstrate that m/z shifts in optimized cellular MALDI-MS protein fingerprints can be used for label-free quantification of hyperacetylated histones. Since MS fingerprints monitor the proximal effect of HDAC inhibition by drugs, they provide a suitable measure of target engagement in cells without the need for cell extraction, histone purification, digestion, timeconsuming LC−MS/MS analysis, or expensive acetylationspecific antibodies. The present method complements the quantitative analysis of individual acetylation sites,7 as it provides very fast, label-free, quantitative and automated MS measurements. Moreover, it affords a clear distinction between singly, doubly, and multiply acetylated states, which is inassessible by antibody-based approaches and would require extensive bioinformatics expertise for implementation in LC− MS/MS-based methods. MALDI mass spectrometers with higher resolving power are expected to provide an analogous means for quantification of histone H3 modifications such as mono-, di-, and trimethylation and correspondingly for profiling of other inhibitor classes such as demethylase inhibitors. Tissue MS analysis of proximal drug responses may therefore enable ex vivo testing of multiple drug candidates in tissue culture, analysis of tumor clonality, and resistance to therapy based on topographic maps of drug activity and the development of advanced drug-delivery systems that target a defined subset of previously nonresponsive target cells.

METHODS Reagents. All reagents used in this study were of HLPC grade. Acetonitrile (ACN), trifluoroacetic acid (TFA), and neat ethanol were purchased from Merck (Darmstadt, GER). MilliQ water (ddH 2 O; Millipore) was prepared in-house. Dimethylsulfoxide (DMSO; cell culture grade) was obtained from Sigma. Sinapinic acid (SA; catalog no. 201345), SDHB (catalog no. 209813), MALDI-MS protein calibration standard I (catalog no. 206355) and indium tin oxide (ITO) slides were 4645 | Anal. Chem. 2014, 86, 4642−4647

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conducted at the University Medical Center Mannheim (UMM) or at Heidelberg Pharma GmbH by trained employees after approval by the local animal welfare committee. Target engagement in tumor tissue by panobinostat (dissolved in 5% dextrose/PBS as described elsewhere19) was examined in two tumor mouse models: Transgenic CEA424/SV40 T-antigen (CEA/Tag) mice, a genetic mouse model for spontaneously developing gastric cancers, and the MDA-MB-468 tumor xenograft model. The 13 to 15 week old female CEA/TAG transgenic mice (C57BL6 mice background) and 15 week old MDA-MB-468 tumor xenografts (NRMI nude mice background) received a single intraperitoneal (i.p) dose of vehicle control (N = 2 per group) or 30 mg/kg LBH-589 (N ≥ 2). CEA/TAG mice were treated for 1 h or 4 h and the MDA-MB468 tumor xenografts for 4 h. Following treatment, animals were anaesthetized under an isoflurane atmosphere and sacrificed by cervical dislocation. Target organs were excised, washed with PBS, and immediately slowly frozen by placement above a cold isopentane22 bath in liquid nitrogen and stored at −80 °C until analysis. Isopentane-frozen organs were mounted onto a metal chuck with frozen section compound and cut into 10 μm cryosections using a Leica Biosystems cryostat CM 1950 at a chamber temperature of −19 °C. Cryosections were thaw-mounted onto conductive glass slides precooled to −19 °C. Before matrix application, ITO slides were desiccated overnight under vacuum at room temperature (RT). For protein IMS, a six step washing protocol was used as described elsewhere,23 briefly, first 70% ethanol (EtOH) for 30 s, second 100% EtOH for 30 s, third Carnoy’s fluid24 (60% EtOH/30% chloroform/ 10% acetic acid v/v/v) for 2 min, fourth 100% EtOH for 30 s, fifth ddH2O for 30 s, and sixth 100% EtOH for 30 s and dried overnight under vacuum at RT. MALDI Imaging MS of Histone Acetylation ex Vivo. The 60 mg/mL SDHB in ACN/ddH2O/TFA (40/60/1.25 v/ v/v) was applied to tissue sections by spray coating using the following parameters of the SunCollect device (SunChrom, Friedrichsdorf, Germany): First, two initial matrix layers, using a flow-rate of 10 μL/min followed by three layers of 15 μL/min were chosen to ensure a homogeneous matrix layer on tissue slides. A drying step of 3 min was introduced between each cycling step. MALDI-IMS was performed in positive linear mode in the mass to charge range of m/z 4 000−20 000 using the FlexImaging 3.0 (Bruker Daltonics). Prior to analysis, the acquisition method was calibrated using the protein calibration standard I (Bruker Daltonics; catalog no. 206355). A total of 400 laser shots were accumulated per raster spot at a laser width of 100 μm. In addition, baseline-subtraction was performed and images were visualized after total ion count normalization. Statistical Analysis and Visualization. Graph design and EC50 values for three HDACi were calculated from ion intensities of the peak corresponding to nonacetylated histone H4 (m/z 11 306, normalized to vehicle control) and were fitted using a nonlinear regression curve fit (log(inhibitor concentration) vs intensity of response) algorithm by Graphpad Prism 5.0 (GraphPad Software Inc., California, USA). Data are presented as mean ± standard deviation (SD) from at least three independent experiments. MALDI cell fingerprint dynamics were investigated and visualized using Tableau software (version 7.0).



S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at


Corresponding Author

*E-mail: [email protected]. Phone +49 (0)621/292-6802. Fax: +49 (0)621/292-6420. Notes

The authors declare the following competing financial interest(s): S.B. and A.P. are employees of Heidelberg Pharma GmbH. This company funded their work (i.e. testing of HDACi in xenograft animal models).

ACKNOWLEDGMENTS The authors thank Professor Dr. Wolfgang Zimmermann, Munich, for providing the CEA424/SV40 T-antigen (CEA/ Tag) mouse model. This work was supported by the following grants: Baden-Württemberg Ministry of Science and Culture INST 874/2-1 LAGG (to C.H.), “ZAFH ABIMAS” by ZO IV/ Landesstiftung Baden-Württemberg and the European Fund for Regional Development (EFRE; to C.H.), by BMBF FHprofUnt Grant 17 001X 11 (to C.H.), by EU CMST COST Action TD0905: Epigenetics: Bench to Bedside (to B.M. and C.H.). This work was supported by the Perspektivförderung des Landes Baden-Württemberg für das Zentrum für Geriatrische Onkologie und Biologie (ZOBEL, to M.P.E.).


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