The Impact of Chemotherapeutic Agents on Signaling and Epigenetics in Vascular Endothelial Cells

Abstract Background: Molecularly targeted anti-cancer therapeutics have revolutionized cancer treatment. However, cardiovascular toxicity is often a dose limiting side effect of these targeted agents. Vascular toxicities include hypertension, increased cardiovascular ischemic events and increased risk of arterial and venous thrombosis. The mechanisms of vascular toxicities are unclear and may be related to on or off target effects in vascular cells. Hypothesis: We hypothesize that small molecule kinase inhibitors cause off target phosphorylation in vascular endothelial cells some of which result in long-term epigenetic changes in histones and that epigenetic modulators also have long-term impact on vascular cell histone modifications. Methods and Results: To test this hypothesis, we used two novel, global, unbiased, high throughput proteomic assays to determine the phospho-signaling (P100 assay) and histone modification profiles (global chromatin profiling (GCP) assay) of human umbilical vein endothelial cells (HUVEC) treated with 26 drugs. The drug panel included 8 epigenetic modulators (vorinostat, decitabine, JQ1-s, GSK126, UNC 0646, GSK J4, resveratrol, geldanamycin), 9 kinase inhibitors (pazopanib, nilotinib, LY-294002, okadaic acid, tofacitinib, SP600125, losmapimod, ARA014418, staurosporine) and 9 other drugs (tretnoin, tacrolimus, sirolimus, lenalidomide, curcumin, KN-62, pravastatin, rolipram, dexamethasone).The proteomic assays were developed as part of the LINCS (Library of Integrated Network-Based Cellular Signatures) program at the Broad Institute. The P100 assay is a mass spectrometry-based targeted proteomics assay that detects and quantifies a representative set of 100 phosphopeptides that are present in a wide range of cell types and from which 1000 unique phosphosites may be imputed. The global chromatin profiling (GCP) is a novel mass spectrometry assay to simultaneously quantify over 70 histone modifications including phosphorylation, ubiquitination, methylation and acetylation. HUVECs cultured in 100 mm plates were treated in triplicate with each drug. After 3 hours, phosphopeptides were collected for the P100 assay and after 24 hours of treatment, histones were extracted for the GCP assay. For each assay, the peptides were mixed with a set of isotopically labeled internal standards and mass spectrometry was performed to quantify each phospho-peptide or modified histone peptide compared to the universal standard. We successfully treated and harvested cells under all proposed conditions above for both assays and mass spectrometry analysis for P-100 assay is underway and completed for GCP assay.We were able to visualize global changes to the histone epigenome in human endothelial cells in response to multiple pertubations for the first time. We found that most kinase inhibitors cause distinct histone epigenetic profiles in HUVECs, indicating more long term effects on vascular cells in addition to short term effects that are expected due to inhibition of specific kinases. We also compared the histone profile obtained in HUVECs to that of several cancer cell lines treated with the same drugs including MCF7 (breast), A549 (lung), A375 (skin) and PC3 (prostate).Some of these changes in histone marks seen in vascular cells are similar to those seen in cancer cells lines, while others are unique to endothelial cells. As predicted, the epigenetic modulators cause histone epigenetic changes in HUVECs. Interestingly, epigenetic modulators that do not directly inhibit enzymes affecting histone modifications (such DNA methylators and demethylators) also cause distinct histone epigenetic signatures in HUVECs. Conclusions: Small molecule anti-cancer agents alter the post-translational modifications present on histones of endothelial cells. In addition to histone changes expected from known epigenetic modulators, kinase inhibitors also produce distinct histone epigenetic signatures in endothelial cells, possibly the result of long term changes caused by target kinase inhibition. Further studies directed at specific histone epigenetic changes could identify critical signaling pathways in endothelial cells and also identify possible mechanisms of vascular toxicities of targeted agents. Disclosures No relevant conflicts of interest to declare.