When studying the potential of HATi for the treatment

When studying the potential of HATi for the treatment of inflammatory lung diseases, investigating lung tissue specific effects may be particularly relevant since local administration of small molecule inhibitors in lung tissue is possible and is already applied for inhaled glucocorticoids in the treatment of these diseases. It should be noted that lung tissue specific effects of 6-alkylsalicylates on histone acetylation have not been reported before. Interestingly, it has been demonstrated that Ac-CoA levels vary between different tissue types, and this influences activities of HATs and HDACs [2]. This indicates that the effects upon treatments with HATi could differ between different tissues and need to be studied in a tissue specific manner. This study demonstrates the feasibility of studying effects of small molecule inhibitors on histone acetylation specifically in lung tissue using a more accurate mass spectrometry based analysis compared to antibody based techniques, which we believe will have added value for the development of these type of inhibitors towards applications in the treatment of inflammatory lung diseases. Others have not done this using mass spectrometry. In this study, reduction of pro-inflammatory gene rad001 was observed in PCLS upon treatment with the 6-alkylsalicylate MG149, supporting the hypothesis that 6-alkylsalicylates have potential for the treatment of inflammatory lung diseases. In lung tissue, effects of MG149 on pro-inflammatory gene expression have not been reported before. However, results obtained with the 6-alkylsalicylate AA in mouse models of lung inflammation are in line with our findings, as exemplified by decreased cytokine expression and a change in the balance of HAT to HDAC activities [15], [16]. Furthermore, AA was reported to influence the NF-κB pathway in a study by Sung et al using human cancer cell lines. Important findings of this study include that AA suppressed NF-κB activation upon TNF-α or LPS stimulus, and that AA suppressed acetylation of p65 upon TNF-α stimulus [34]. Additionally, for MG149 it is also interesting to note that this inhibitor appears to have selective effects on gene expression, as was shown using a microarray analysis where this inhibitor mainly affected the NF-κB and p53 pathways in MOLP8 and K562 human cancer cell lines [32]. Altogether this indicates that 6-alkylsalicylates influence pro-inflammatory events in cellular systems. Other literature concerning MG149 has also focused on alternative applications. For example, MG149 has been used to study the role of KAT5 in chromosome segregation [35]. This indicates that next to applications where 6-alkylsalicylates like MG149 could be used as a drug, these small molecules can also be used as a tool to study the role of MYST type HATs in fundamental cellular processes. In summary, our findings indicate that the 6 alkylsalicylate MG149 inhibits histone acetylation and expression of several pro-inflammatory genes in murine precision cut lung slices. This sets the stage for further development of this compound class towards applications in the treatment of inflammatory lung diseases such as asthma and COPD. Future directions should focus on improving Ki values and selectivity for the HAT enzymes for these compounds.
Acknowledgements We acknowledge the EU-COST action TD0905 ‘epigenetics from bench to bedside’ for funding a short term scientific mission of TvdB to the group of Axel Imhof, Ludwig Maximilians University, Munich. We acknowledge the EU-COST action CM1406 ‘epigenetic chemical biology’ for building a scientific network. We acknowledge the European Union for funding this project by an ERC starting grant to FJD (309782). Further support was obtained from The Netherlands Organisation of Scientific Research (NWO) by a VIDI grant to FJD (723.012.005). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We acknowledge Prof. Dr. Reinoud Gosens (Department of Molecular Pharmacology, University of Groningen) for support with ex vivo experiments regarding precision-cut lung slices. We acknowledge Andries Heida for support with experiments regarding histone acetylation in RAW264.7 cells.