Accepted Manuscript Evaluation of [11C]KB631 as a PET tracer for in vivo visualisation of HDAC6 in B16·F10 melanoma Koen Vermeulen, Muneer Ahamed, Kaat Luyten, Guy Bormans PII: S0969-8051(19)30083-6 DOI: https://doi.org/10.1016/j.nucmedbio.2019.05.004 Reference: NMB 8069 To appear in: Nuclear Medicine and Biology Received date: 2 April 2019 Revised date: 9 May 2019 Accepted date: 14 May 2019 Please cite this article as: K. Vermeulen, M. Ahamed, K. Luyten, et al., Evaluation of [11C]KB631 as a PET tracer for in vivo visualisation of HDAC6 in B16·F10 melanoma, Nuclear Medicine and Biology, https://doi.org/10.1016/j.nucmedbio.2019.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Evaluation of [11C]KB631 as a PET tracer for in vivo visualisation of HDAC6 in B16.F10 melanoma Abbreviated title: [11C]KB631 for HDAC6 melanoma visualisation Koen Vermeulen1, Muneer Ahamed2, Kaat Luyten1, 3 Guy Bormans1 1 Laboratory for Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, KU Leuven, Leuven, Belgium 2 Centre for Advanced Imaging, University of Queensland, Brisbane, Australia 3 Switch Laboratory, VIB-KU Leuven Center for Brain & Disease Research, KU Leuven, Leuven, Belgium Corresponding author: Prof. Dr. Guy Bormans. Email: [email protected]; Tel: +3216330447; Herestraat 49, box 821, O&N II, Campus Gasthuisberg, 3000 Leuven, Belgium Keywords: HDAC6, KB631, B16.F10 melanoma, carbon-11 Abstract Introduction: HDAC6, a structural and functional distinct member of the HDAC-family, shows great promise as a target to treat several cancers and neurodegenerative diseases. Several clinical trials are evaluating HDAC6 inhibitors in solid tumours and haematological malignancies, but so far no HDAC6 inhibitor has received marketing authorisation. The availability of an HDAC6- specific PET tracerACCEPTED can potentially aid in cancer MANUSCRIPT diagnosis, select patients for HDAC6 inhibitor treatment and accelerate HDAC6 drug development. We have evaluated the HDAC6 PET tracer [11C]KB631, in vitro and in vivo in B16.F10 melanoma inoculated mice. Methods: The radiosynthesis of [11C]KB631 was optimized. In vitro binding specificity was evaluated by autoradiography studies on rodent brain, B16.F10 melanoma and PC3 prostate 1 ACCEPTED MANUSCRIPT carcinoma cryosections. Biodistribution and quantification of plasma radio-metabolites was determined in NMRI-mice in control conditions and after blocking with KB631, Ricolinostat and SAHA. Tracer tumour uptake was evaluated in B16.F10 melanoma inoculated C57BL/6 mice. Results: In vitro autoradiography studies showed HDAC6-selective binding to rodent brain, B16.F10 melanoma and PC3 prostate carcinoma tissue slices. Tracer binding in several organs of interest could be partially blocked in NMRI-mice pre-treated with KB631, Ricolinostat or SAHA, indicating specific tracer binding. A biodistribution and 90-min dynamic µPET study on B16.F10 melanoma mice, pre-treated with vehicle or Ricolinostat (50 mg/kg), indicated HDAC6-specific tumour uptake. Conclusions: [11C]KB631 shows HDAC6-selective binding in mouse B16.F10 melanoma tumours in vitro and in vivo. [11C]KB631 PET can be used for in vivo investigation of the expression of HDAC6 in tumours. Advances in Knowledge: [11C]KB631 shows increased expression of HDAC6 in mouse B16.F10 melanoma tumours and can be used to visualise target engagement of HDAC6 inhibitors. 1. Introduction The alternate acetylation state of evolutionarily conserved lysine residues located at the N- terminal tails of histoneACCEPTEDs contributes to the general MANUSCRIPT transcriptional regulation of underlying genes. This process, controlled by the opposing actions of histone acetyl transferases (HATs) and histone deacetylases (HDACs), is part of a broader network of epigenetic post translational modifications (PTMs) [1], contributing to the histone homeostasis which in turn influences 2 ACCEPTED MANUSCRIPT cellular homeostasis. Aberrant levels of HAT/HDAC can lead to various pathologies, ranging from oncological malignancies to cardiac diseases and even neurophysiological anomalies [2,3]. Currently 18 HDAC-isoforms are known, divided into 4 classes based on their sequence homology to yeast HDAC: Class I (HDAC1, 2, 3, 8), class II (HDAC4, 5, 6, 7, 9, 10), class III (sirtuins) and class IV (HDAC11). The various classes differ in protein structure, substrate specificity, subcellular localization and tissue expression patterns [3]. All classes except class III are known to use Zn2+ as a catalysing agent to facilitate the deacetylation reaction. Class III HDACs use nicotinamide adenine dinucleotide (NAD+) to hydrolyse the acetyl-moiety [4]. Observation of the disruption of HAT/HDAC homeostasis in several malignancies led to the identification of HDAC as a drug target. However, because of the high similarity between different HDACs, e.g. HDAC1 and 2 have 85% sequence homology, pan-inhibition of HDACs can lead to serious adverse effects. Consequently, research has focussed on developing isoform- selective inhibitors. For HDAC3, 6 and 8, selective inhibitors have already been reported [5]. Inhibitors of Zn2+-dependent HDACs typically, contain (1) a ‘cap’ group, which interacts with the surface of the catalytic pocket of the HDAC enzyme, (2) a linker, which can contain different aromatic rings and/or alkyl chains, connected to (3) the Zn2+-binding moiety. Chelation of the Zn2+-ion can be accomplished with multiple functional groups, such as carboxylic acids, benzamides, thiol groups or hydroxamates [6]. FDA approved HDAC inhibitors, depicted in Fig. ® 1A include panACCEPTED-HDAC inhibitors Vorinostat MANUSCRIPT (Zolinza also known as suberoylanilide hydroxamic acid (SAHA)) [7] and Romidepsin, (Istodax®) [8] both used for the treatment of cutaneous T-cell lymphoma (CTCL). In addition, Belinostat (Beleodaq®) [9], applied for the treatment of peripheral T-cell lymphoma (PTCL) and Panobinostat (Farydak®) [7] used to combat multiple myeloma (MM) are available on the market. IC50 values are given in Table 1. 3 ACCEPTED MANUSCRIPT HDAC6 is a unique HDAC isoform as it contains two homologous catalytic deacetylase domains. Both domains function individually and participate in the global deacetylase activity of the enzyme. Contrary to class I HDACs, HDAC6 is predominantly localized in the cytoplasm and subsequently targets cytosolic acetylated proteins. Mainly α-tubulin, heat shock protein 90 (Hsp90) and cortactin are targeted and deacetylated by HDAC6. Deacetylation of α-tubulin and cortactin is implied in cytoskeleton dynamics and cell motility and deacetylation of the molecular chaperone Hsp90 is necessary to activate a cellular response to misfolded proteins and stress [10,11]. Expression of HDAC6 was reported in several organs, including: heart, liver, kidney, brain and pancreas [12]. HDAC6 is a key regulator of multiple cellular signalling and downstream transduction pathways. The regulation of different cellular processes including cell migration and the degradation of misfolded proteins, are not solely attributed to the deacetylation process, as HDAC6 also contains a C-terminal, zinc finger containing, ubiquitin binding domain (BUZ-domain) that is equally important in the control of these processes [13]. The BUZ-domain is able to bind free ubiquitin or ubiquitinated proteins destined for proteasomal degradation [14]. Misfolded or damaged proteins will be marked with a poly-ubiquitin tag after which different degradation pathways can be followed. The most prominent degradation route is the transport of misfolded proteins to the proteasome. However, if the proteasome becomes oversaturated or inhibited, anotherACCEPTED process is required to remove MANUSCRIPT the cytotoxic, misfolded or damaged proteins. This process, known as the aggresome-autophagy pathway, is initially cytoprotective and induces accelerated degradation of mutant proteins. The pathway is initiated and regulated by high affinity binding of HDAC6 to poly-ubiquitinated proteins [15,16]. HDAC6 was found to play a role in cancer and neurodegenerative diseases [12,17]. HDAC6 is overexpressed in a variety of human cancers and is required for oncogenic cell transformation 4 ACCEPTED MANUSCRIPT [12,18]. The HDAC6-gene is estrogen-regulated and increased HDAC6 mRNA and protein expression was observed in estrogen receptor α-positive breast cancer MCF-7 cells treated with estradiol. In the same study, a fourfold increase in cell motility and cellular morphological changes caused by the deacetylation of α-tubulin was observed [19]. Expression levels of HDAC6 were also increased in ovarian cancer, specifically in non-benign tumours, where HDAC6 potentially can be used as prognostic marker [20]. In prostate and kidney cancer, upregulation of HDAC6 was mediated by oncogenic retrovirus-associated DNA sequences (Ras) [21]. Overexpression was also found in oral squamous cell carcinoma, melanoma and several hematopoietic cancers [22–24]. Importantly, it has been observed that HDAC6 inhibition sensitises cancerous cells to chemotherapeutics, but not normal untransformed cells [25]. Deletion or