Unexpected selectivity of histone deacetylase inhibitors in live MDA-MD-231 triple-negative breast cancer cells

While inhibition of histone deacetylases (HDAC) is a promising strategy for the treatment of cancer, HDACs do play essential roles in normal cellular function, limiting their use as therapeutic targets. Increasing evidence shows that each of the HDAC isoforms carry out unique biological functions; therefore, by inhibiting a single isoform, one could regulate a focused subset of genes and achieve the desired therapeutic effect and lower toxicity. To investigate this aspect, many isoform-selective inhibitors have been synthesized, characterized for selectivity against recombinant HDACs in biochemical assays, and tested for anti-cancer activity. Despite significant progress, the actual engagement of HDAC isoforms is poorly characterized since methods to determine selectivity of HDAC inhibitors in live cells with unperturbed epigenetic machinery and in vivo are missing. Recently, using a novel photoreactive HDAC probe (PRP), we demonstrated that its isoform selectivity in live breast cancer cells is cell-type dependent, differs from that determined in the biochemical assays, and, in the case of HDAC3, is regulated by phosphorylation by c-Jun N-terminal kinase (JNK). Similar cell-type dependent variability in HDAC isoform selectivity was also observed for other HDAC isoforms. In this report, we extended our photolabeling approach to characterize HDAC isoform selectivity of the commonly used in pre-clinical and clinical research non-selective and selective inhibitors: SAHA, panobinostat, pimelic diphenylamide 106, PCI-34051, and TMP-269. Seven novel PRPs based on the chemical scaffolds of these HDAC inhibitors were synthesized and tested in the biochemical assay and the cell-based photolabeling assay in live triple negative breast cancer cells MDA-MB-231. We found that the cell-based profiles of these PRPs were skewed toward inhibition of HDAC3 irrespective of their biochemical selectivity, chemotypes, or isoform abundance, suggesting that the actual HDAC isoform selectivity and target engagement in live cells is different from that perceived based on the results of the biochemical assay. These studies suggest that a substantial correction in our understanding of the role HDAC isoforms and their inhibitors play in live cells in culture and in vivo may be necessary. Further expansion of these studies to other types of cells and animal tissues ex vivo and in vivo as well as identification of the mechanisms responsible for modulation of HDAC isoform selectivity and target engagement are in progress. Citation Format: Shaimaa M. Aboukhatwa, Thomas W. Hanigan, Jayaprakash Neerasa, Taha Y. Taha, Nathan D. Brown, Eman E. El-Bastawissy, Mohamed A. Elkersh, Tarek F. El-Moselhy, Jonna Frasor, Pavel A. Petukhov. Unexpected selectivity of histone deacetylase inhibitors in live MDA-MD-231 triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5801.