The Library of Integrated Network-based Cellular Signatures (LINCS) Program aims to create a network-based understanding of biology by cataloging changes in gene expression and other cellular processes that occur when cells are exposed to a variety of perturbing agents. The goal of this project is to test the hypothesis that modulation of phosphorylation-mediated signaling events in response to perturbations can establish new cellular states by altering their epigenetic landscape. We have performed mass spectrometry (MS)-based proteomic assays that target quantitative readouts of phospho-signaling and chromatin modifications in cellular models, following perturbation by compounds with varying mechanisms of action. We have employed 128 compounds and treated 5 cancer and 2 neuronal cell lines. The resulting data collected and tools created have been contributed to the LINCS program for the purpose of making connections among disparate perturbations through phosphoproteomics and chromatin modification signatures in concert with other available data types. The data is publicly available on PanoramaPublic and can be queried using Clue (Broad Institute). Analysis of our data will inform novel therapeutic opportunities and synergies, as dysregulation of phospho-signaling and epigenetic systems are two of the most common molecular etiologies identified in a growing number of diseases.

Detailed GCP protocols can be found in Creech et al., 2015, Methods and online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=sops. Cells were washed with PBS and frozen immediately after drug treatment (24 hrs). Nuclei were extracted from cells using the classic low-detergent lysis method (0.3% NP-40). Histones were extracted from the nuclei with 0.4 N H2SO4 and solubilized histones were precipitated from solution using 20% w/v trichloroacetic acid. Following isolation, 10 μg of histone proteins were chemically propionylated with NHS-propionate, cleaned using reversed phase SPE cartridges, and digested overnight with trypsin. Peptides were then propionylated again to block nascent N-termini. Finally, a cocktail of isotopically-labeled heavy peptides were spiked into each sample to allow for quantitation and cross-sample comparisons. Samples were analyzed using liquid-chromatography mass spectrometry. Samples were injected using a targeted acquisition method to monitor for the presence of defined combinatorial forms of modified histone peptides. Results were analyzed independently using Skyline software package (MacLean et al., 2014) as described in (Creech et al. 2015).

Drug signatures were profiled in various cancer cell lines (A375, YAPC, A549, MCF7, and PC3) and neuronal cell models (Astrocytes & NPCs). Drugs were grouped into four tranches: epigenetically active, neuroactive, kinase inhibitors and cardiotoxic compounds. A detailed description of their mechanism of action can be found online at https://panoramaweb.org/wiki/LINCS/Overview%20Information/page.view?name=LINCS%20PCCSE%20Overview and https://clue.io/touchstone. Drug stocks were dissolved in DMSO for treatment in cell line media and cells were treated in 6 well plates for 24 hours. For cell harvesting, cells from 1-2 wells of a 6 well plate were scraped in PBS and combined per sample. All drug perturbations occurred in triplicates.