Label-Free in Situ Monitoring of Histone Deacetylase Drug Target Engagement by Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Biotyping and Imaging

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 gastrointestinal 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.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer

“…MALDI-MSI [32] confirmed rapid in situ distribution and metabolism [40] of fasudil and its metabolite hydroxyfasudil in liver and normal stomach tissue of wild-type (WT) mice and in gastric tumor tissue of transgenic mice. Unexpectedly, the highest ion intensities for the two compounds were located in the non-malignant upper part of the stomach (corpus), whereas lower signal intensities were found in the tumor regions of the lower stomach (pylorus).…”

“…Because RHOA mutations have been identified as major oncogenic drivers of the diffuse type of GC [3], [4], [5], we tested the hypothesis that inhibition of the RHO-signaling pathway shall reduce gastric tumor growth in vivo . We resorted to a transgenic mouse model of GC [27], where the viral oncogene SV40 large T-antigen is expressed as a transgene under the human CEA promoter specifically in the lower stomach (pylorus) of the mice, and which has been successfully subjected to pharmacological intervention by us before [32], [43], [44]. This tumor is characterized by early onset (within 4 weeks of age), a high proliferation index and up-regulation of stem cell (Wnt/Notch) [45] and neuroendocrine differentiation signatures [28] culminating in a small cell carcinoma of the lower part of the mouse stomach, the pylorus.…”

“…Animals were treated for 10, 30, or 60 min before being sacrificed. For imaging studies [32], [33], [34], stomach and liver tissues were dissected, washed with PBS and immediately frozen in cryomolds above a pre-cooled isopentane bath in liquid N 2 . Organs were stored at −80°C until analysis.…”

Scores for standardization of on-tissue digestion of formalin-fixed paraffin-embedded tissue in MALDI-MS imaging

“…This technology enables in-depth molecular analysis of tissue slices with increasingly high spatial resolution. It generates unique representations of the distribution of lipids, metabolites, peptides, proteins pharmaceuticals or drug responses [3][4][5][6]. Currently MALDI MSI is primarily performed on frozen tissue samples, but an increasing number of studies are conducted on formalin-fixed paraffin-embedded (FFPE) material kept in pathology facilities for long-term storage.…”

Two-Photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement

“…the ability of a drug to interact with a receptor, has been so far measured indirectly by monitoring cellular response following activation of the drug downstream signal, but recently new approaches have been developed capable of directly measuring drug binding in cells and in vivo . Drug affinity responsive target stability (DARTS)[3], positron emission tomography [4, 5], and mass spectroscopy [6] help determining the degree of drug interaction with a protein, the degree of drug accumulation in a tumor through drug radiolabeling, or to determine drug target engagement in situ through desorption of the sample and analysis. Also cellular thermal shift assay (CETSA) [7], has extended target engagement measurements to cells and soluble protein fractions extracted from cell lysates.…”