Selective pharmacological inhibitors of HDAC6 reveal biochemical activity but functional tolerance in cancer models Yves Depetter 1,2,3 , Silke Geurs 1 , Rob De Vreese 1 , Sophie Goethals 2 , Elien Vandoorn 2 , Alien Laevens 2 , Jonas Steenbrugge 4 , Evelyne Meyer 3,4 , Pascal de Tullio 5 , Marc Bracke 2,3 , Matthias D’hooghe 1 and Olivier De Wever 2,3 1 SynBioC Research Group, Department of Green Chemistry and Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 2 Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium 3 Cancer Research Institute Ghent (CRIG), Ghent, Belgium 4 Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 5 Center for Interdisciplinary Research on Medicines (CIRM), Metabolomics Group, Université de Liège, Liège, Belgium Our study investigates the biochemical and functional impact of selective histone deacetylase 6 (HDAC6) inhibitors, a promising class of novel therapeutics, in several cancer models. Selective HDAC6 inhibitors (Tubathian A, Tubastatin A, Tubacin and Ricolinostat) and a non-selective HDAC inhibitor (Vorinostat) were evaluated on cancer cell lines derived from multiple tumour types in both an in vitro and in vivo setting as potential cancer therapeutics. Selective HDAC6 inhibitors resulted in α-tubulin acetylation with no impact on histone acetylation but failed to show any anti-cancer properties. Only the use of high concentrations of selective HDAC6 inhibitors resulted in co-inhibition of other HDAC enzymes and consequently in reduced growth, migratory and/or invasive activity of cancer cells in vitro as well as in vivo. The specificity of HDAC6 inhibition was confirmed using a CRISPR/Cas9 knockout cell line. Our results suggest that selective HDAC6 inhibitors may fall short as potential single agent anti-cancer drugs and prove that many previous data regarding this promising class of compounds need to be interpreted with great care due to their use in high concentrations resulting in low selectivity and potential off-target effects. Introduction Histone deacetylases (HDACs) are a class of enzymes respon- sible for altering the acetylation status of target proteins by removing acetyl groups from side-chain acetylated lysine (K)- residues. 1 There are four classes of HDACs, each containing different isozymes, divided according to their homology to their yeast counterparts, cellular localization and substrate specificity: the zinc-dependent class I (HDAC1-3 and 8), class II (HDAC4-7 and 9–10) and class IV (HDAC11) and the nic- otinamide adenine dinucleotide (NAD + )-dependent class III or sirtuins. 2,3 Class I HDACs reside predominantly in the nucleus where their main substrate, histones, can be found. Class II enzymes can shuttle between the nucleus and cyto- plasm and HDAC6 is located almost exclusively in the cyto- plasm. HDAC11 bears the closest resemblance to HDAC3 and 8 and resides mainly within the nucleus. 2,3 Sirtuins differ from the classical HDAC family, requiring NAD + for their activity instead of Zn 2+ . Since HDACs are often overexpressed in cancer and are believed to play an essential role in carcinogenesis and cancer progression, they are promoted as promising therapeutic tar- gets. Capitalizing on this, numerous HDAC inhibitors (HDACi) have been designed as clinical agents, leading to the FDA approval of four HDACi for the treatment of specific types of T-cell lymphoma and multiple myeloma: Vorinostat (SAHA), Romidepsin, Panobinostat and Belinostat. HDACi act through complexation of the essential Zn-kation, present in the catalytic site of the enzymes, thus inhibiting the deace- tylase activity. They assert their biological effect by inducing apoptosis or cell-cycle arrest, reducing angiogenesis and/or metastasis and enhancing anti-tumour immunity. 4–6 On the downside, the majority of HDACi are non-selective, implying that they inhibit multiple HDAC isozymes, and display toxic side effects due to their broad activity. 7 In light of this, recent research focusses on the development and implementation of isoform-selective HDACi. Key words: histone deacetylase 6, inhibition, Tubathian A, Tubastatin A, tumour Additional Supporting Information may be found in the online version of this article. Conflict of interest: The authors declare no conflict of interest. DOI: 10.1002/ijc.32169 History: Received 12 Oct 2018; Accepted 22 Jan 2019; Online 29 Jan 2019; Correspondence to: Olivier De Wever, Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, B-9000 Ghent, Belgium, Tel.: +32-9-3323073, Fax.: +32-9-3324991, E-mail: olivier.dewever@ugent.be International Journal of Cancer IJC Int. J. Cancer: 145, 735–747 (2019) © 2019 UICC Cancer Therapy and Prevention