HDAC6

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Perspective

Old but Gold: Tracking the New Guise of Histone Deacetylase 6 (HDAC6) Enzyme as a Biomarker and Therapeutic Target in Rare Diseases Margherita Brindisi, A. Prasanth Saraswati, Simone Brogi, Sandra Gemma, Stefania Butini, and Giuseppe Campiani J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00924 • Publication Date (Web): 15 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Journal of Medicinal Chemistry

Old but Gold: Tracking the New Guise of Histone Deacetylase 6 (HDAC6) Enzyme as a Biomarker and Therapeutic Target in Rare Diseases Margherita Brindisi≠, A. Prasanth Saraswati§, Simone Brogi¥, Sandra Gemma§, Stefania Butini§, Giuseppe Campiani§,*

≠Department

of Pharmacy, Department of Excellence 2018-2022, University of Naples Federico

II, Via D Montesano 49, I-80131 Naples, Italy §Department

of Biotechnology, Chemistry and Pharmacy, Department of Excellence 2018-2022,

University of Siena, via Aldo Moro 2, 53100, Siena, Italy ¥Department

of Pharmacy, University of Pisa, via Bonanno 6, 56126, Pisa, Italy

KEYWORDS: Epigenetics, HDAC enzymes, HDAC6, rare diseases, Rett syndrome, CharcotMarie Tooth disease, Inherited retinal disorders, idiopathic pulmonary fibrosis

Abstract Epigenetic regulation orchestrates many cellular processes and greatly influences key disease mechanisms. Histone deacetylase (HDAC) enzymes play a crucial role either as biomarkers or therapeutic targets owing to their involvement in specific pathophysiological pathways. Beyond their well-characterized role as histone modifiers, HDACs also interact with several non-histone substrates and their increased expression has been highlighted in specific diseases. HDAC6

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isoform, due to its unique cytoplasmic localization, modulates the acetylation status of tubulin, HSP90, TGF-β and peroxiredoxins. HDAC6 also exerts non-catalytic activities, through its interaction with ubiquitin. Both catalytic and non-catalytic functions of HDACs are being actively studied in the field of specific rare disorders, beyond the well-established role in carcinogenesis. This perspective outlines the application of HDAC(6) inhibitors in rare diseases, such as Rett syndrome, inherited retinal disorders, idiopathic pulmonary fibrosis and Charcot-Marie-Tooth disease, highlighting their therapeutic potential as innovative and targeted disease-modifying agents.

1. Introduction Epigenetics is a natural progression of genetics and represents one of the most attractive research areas in medicine and modern biology; it describes the influence of genetic processes on development and studies the inherited modifications in gene expression which do not encompass alterations in the original DNA sequence, namely the changes in the phenotype not involving any corresponding changes in genotype.1-4 Epigenetic modifications occur naturally but they could also be favoured by environment, age, nutrients, disease states and lifestyle. Epigenomic profiling has allowed defining the critical DNA control elements, such as gene promoters and enhancers. In combination with DNA sequence analysis, epigenetic research has provided deep insights into disease pathways. Also, the successful completion of the Human Genome Project prompted an effective investigation of epigenetic mechanisms involving scientists from various disciplines. Epigenetics comprises a series of mechanisms by which DNA and its associated proteins, or other crucial protein substrates, get chemically or structurally altered. Chemical alteration includes


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modifications of DNA and histones, whereas, structural changes occur via chromatin remodelling and inter/intrachromosomal DNA-DNA interactions. The typical epigenetic modifications include three main processes, such as histone modifications, DNA methylation, and gene silencing related to non-coding RNA (ncRNA). DNA methylation involves the enzymatic insertion of a methyl (CH3) group on the cytosine rings with both spatial and temporal precision, while histone posttranslational modifications (PTMs) include specific methylation, acetylation, phosphorylation, sumoylation and ubiquitination.5 2. Epigenetics in rare diseases While genetic mutations are irreversible, epigenetic modifications are reversible in nature and may be modulated by targeting enzymes or other factors involved in their regulation.6 Many of these enzymes mediating epigenetic processes, such as histone deacetylase (HDAC) and DNA methyltransferase (DNMT), are usually deregulated in a variety of disease conditions. Approval of anticancer epigenetic drugs, such as the potent HDAC inhibitors vorinostat (SAHA, 1) and romidepsin (2) (Figure 1 and Table 2) has inspired the development of drugs targeting the epigenetic machinery for other disorders, especially neurodegenerative disorders and rare diseases. Rare diseases often termed as orphan diseases, are health conditions which affect a small number of individuals. However, the cut-off number for a disease to be defined as rare differs from region to region.7 To date, 5,000-8,000 distinct rare diseases have been documented, and new rare disorders constantly appear in the scientific literature.8 The vast majority of rare diseases still lack therapeutic solutions partially due to the limited understanding of their pathological mechanisms, but also due to the lack of druggable targets. Owing to the low incidence of rare disorders, the quest for potential therapeutic options from both academia and industry is relatively slow and



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unfruitful. Most rare diseases are genetic in nature which could be either life-threatening or debilitating. However, some rare disorders are characterized by an epigenetic basis. In particular, the nervous system due to its highly complex structure is vulnerable to epigenetic changes; accordingly, several aberrations in the epigenetic machinery have been associated to the insurgence of a number of mental disorders. For example, Rett syndrome (RTT) involves mutations of genes associated to DNA methylation in the methyl-binding domain protein, such as the methyl-CpG binding protein 2 (MeCP2).

9-11

Besides cancerous and central nervous system

(CNS)-related disorders, epigenetic changes are crucial factors also in the progress of immunological and inflammatory disorders. Among them, idiopathic pulmonary fibrosis (IPF) is one such case characterized by progressive lung damage, causing the loss of pulmonary function, respiratory failure and death. Epigenetic mechanisms have been associated to the pathogenesis of IPF, thus playing a key role for the identification of either crucial biomarkers or potential therapeutic targets. 9, 12, 13 Moreover, the contribution of epigenetic mechanisms has been recently unveiled in other rare disorders with limited therapeutic options so far, namely inherited retinal disorders (IRDs)14 and Charcot-Marie Tooth (CMT) disease.15

3. HDAC enzymes and HDAC6 isoform Acetylation of the -amino group of lysine residues is a dynamic PTM finely tuned by the opposite action of lysine acetyltransferases (KATs) and HDACs.16,

17

Hyperacetylation is associated to

transcriptionally active genes and to the decondensed chromatin status, therefore maintaining the unfolded structure of transcribed nucleosome.18,

19

HDACs remove the acetyl groups from

histones, resulting in decreased gene transcription. Currently, 18 HDAC isoforms have been recognized in mammalian cells, and they are clustered based on their homology to yeast HDACs,


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into four classes. HDACs belonging to classes I, II, and IV are zinc-dependent, while class III HDACs are NAD+ dependent. HDAC isoforms 1, 2, 3 and 8 (class I) are expressed in the nucleus of the cells of all tissues and share homology with yeast HDAC RDP3. While HDAC isoforms 1 and 2 are mostly nuclear, isoforms 3 and 8 are able to shuttle in and out of the cell nucleus.20 Some of their key substrates include steroid receptors, tumor suppressors and transcription factors. Class II HDACs show homology to yeast HDAC HDA1 and perform tissue-specific functions, including deacetylation of non-histone substrates. Class II HDACs are further clustered into Class IIA (HDAC isoforms 4, 5, 7 and 9) and Class IIB (HDAC isoforms 6 and HDAC10). Class IV HDACs, only composed by HDAC isoform 11, shares homology with Class I and II enzymes. Class III HDACs, named sirtuins (SIRT isoforms 1-7), are homologous to yeast sirtuin protein Sir2. They are broadly expressed in human tissues and are involved in the regulation of DNA repair, metabolism, oxidative stress, and ageing.21, 22 Among epigenetic regulation processes, HDAC inhibition holds an enormous therapeutic potential23 and it is known to exert pleiotropic effects at both cellular as well as systemic levels Mounting evidence also shows that HDAC inhibition also possesses anti-inflammatory properties.24 HDAC inhibitors can be clustered based on their structure into four large classes: hydroxamates, benzamides, cyclic peptides, and short-chain fatty acids. The three hydroxamatebased HDAC inhibitors approved by FDA, namely SAHA, belinostat (3), and panobinostat (4), behave as pan-inhibitors (Figure 1). The fourth HDAC inhibitor, FK228 (romidepsin, 2, Figure 1), is a Class I selective cyclic peptide (Figure 1 and Table 2). All of them have been approved to treat cancers.



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O

O O O

H N

HN N H

O

OH

1, SAHA (Vorinostat)

S

HN S

O

HN

N H O 2, Romidepsin

O

O

O O S N H

N H

3, Belinostat

OH

HN

HN OH

Me

O

NH 4, Panobinostat

Figure 1. FDA approved HDAC inhibitors 1-4. Despite the successful approvals of the four HDAC inhibitors by FDA, their non-selective HDAC inhibition profile represents a significant drawback, being associated to the occurrence of several side effects. In this regard, increasing investigations are being focused on the identification of isoform-selective HDAC inhibitors as drugs for epigenetic therapy.25 Due to the unique cytoplasmic localization of HDAC6, and its diverse protein substrates, selective HDAC6 inhibitors are increasingly emerging as potential therapeutic agents for cancer, neurodegenerative diseases, and immunological disorders.26, 27 3.1 Structure of HDAC6. HDAC6 isoform was identified in 1999 and displays a unique full duplication of the class I/II homology domain. The human isoform (Figure 2) contains 1255 amino acids and displays two deacetylase binding domains namely DD1 and DD2.28 Besides this, it also has SE14 (Ser-Glu tetra decapeptide repeat domain) and C-terminus hydrolase-like zinc finger domain for ubiquitin binding (ZnF-UBP). At the N-terminal, it has a conserved nuclear export signal (NES) sequence.29 Experimental data using purified HDAC6 indicated that DD2 is responsible for its deacetylation activity on histones and α-tubulin.30 Moreover, HDAC6 enzyme also displays peptide moieties which are necessary for its cytoplasmic localization. In response to


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cellular signals, HDAC6 shuttles between the cytoplasm and the nucleus, wherein, NES and SE14 are responsible for the stability and cytoplasmic localization of the enzyme. HDAC6 also contains a unique zinc finger motif in its C-terminus end, with conserved cysteine- and histidine-rich regions. ZnF-UBP specifically binds to both mono- and poly-ubiquitin chains.31, 32

Figure 2. HDAC6 structure: domain organization and functions. Not only HDAC6 displays a variety of non-histone substrates, but it also interacts with several proteins regulating its deacetylase activity through protein-protein interaction.33 Table 1 summarizes HDAC6 substrates and interacting proteins, while next sub-paragraphs will analyze in detail substrates or interaction specifically involved in the pathomechanisms of the rare disorders herein discussed.



Table 1. HDAC6 substrates and proteins regulating HDAC6 deacetylase activity through protein-protein interactions. Protein -Tubulin34 Cortactin35 HSP9036 -Catenin37 Peroxiredoxin38 Survivin39 Ubiquitin31 Tau40 IIp4541 EGFR42

Type of Interaction with HDAC6 Substrate Substrate Substrate Substrate Substrate Substrate Protein-protein Protein-protein Protein-protein Protein-protein

3.2 HDAC6 and α-tubulin. Tubulin was the first acknowledged HDAC6 protein substrate. The acetylation of α-tubulin at lysine 40 is a common process in the microtubules. Regulation of microtubule function and stability is strongly dependent upon acetylation status of α-tubulin.43 Chemotactic cell movement and tubulin hypoacetylation are promoted by increased expression of HDAC6 in cells (Figure 3).44 Acetylated α-tubulin regulates motor-based trafficking and tubulin binding to kinesin-1, which is required for transporting cargos along with the microtubule network. 3.3 HDAC6 and HSP90. Heat shock protein 90 (HSP90) was identified as the second substrate for HDAC6.45 HDAC6 inhibition or knockdown increase acetylated HSP90 levels and lead to loss of its chaperone activity, by dissociation from co-chaperone p23. This event halts the maturation of the glucocorticoid receptor (GR). HDAC6 has been found to exist in a complex with HSP90 and was seen to be capable of detecting ubiquitinated cell aggregates (Figure 3). The dissociation of HDAC6- heat shock transcription factor 1-(HSF1)-HSP90 complex causes the activation of HSF1, thus prompting the expression of important cellular chaperones.46 These evidences highlight the


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crucial regulatory role of HDAC6 in the cellular response to the event of cytotoxic protein aggregate formation.

Figure 3. Interaction of HDAC6 with α-tubulin and HSP90. 3.4 HDAC6 and ubiquitin. The fact that HDAC6 binds ubiquitin an co-localizes with ubiquitinated aggresomes supports the existence of ubiquitin-dependent activities for this enzyme.31, 32, 47 The ubiquitin binding-activity mediates the shuttling of ubiquitinated aggregates to the aggresomes along microtubules.47 Aggresomes represent the endpoint of microtubule-mediated transport of misfolded polyubiquitinated proteins and HDAC6 plays a vital role in their degradation.48 HDAC6 binds to ubiquitin with a high affinity (Kd = 60 nM), thus stabilizing polyubiquitin chains and inhibiting degradation of ubiquitinated proteins via ubiquitin-proteasome system. Moreover, HDAC6 binds phospholipase A2 inactivating protein (PLAP) and p97/valosin containing protein (p97/VCP), two proteins involved in the control of ubiquitinated substrates.49 p97/VCP is an AAAATPase chaperone with a crucial role in the control of ubiquitin-dependent endoplasmic reticulumassociated degradation. Besides this, it is able to dissociate the complex between HDAC6 and



ubiquitin and to counteract the accumulation of polyubiquitinated proteins promoted by HDAC6.49, 50 The equilibrium between the cellular levels of HDAC6 and p97/VCP is, therefore, a key aspect influencing the fate of polyubiquitinated proteins, which could either undergo proteasomal degradation by p97/VCP or be sequestered into the aggresome (Figure 4). 3.5 HDAC6 and tau. Tau proteins, comprising of six distinct tau forms located in the brain, normally act by binding and stabilizing neural microtubules. Binding of tau to microtubules is regulated by the serine/threonine phosphorylation status, mostly in the proline-rich regions of tau, and also by lysine acetylation directly in the microtubule-binding repeat region. HDAC6 binds tau in in vitro settings and in tissues derived from human brain. In healthy tissues, HDAC6 actively binds to tau and shuttles it for targeted sequestration or degradation. Moreover, HDAC6 is also seen to play a role in the tau hyperphosphorylation process. Tau phosphorylation impairs the binding and stabilizing the function of tau, resulting in self-assembly and aggregation, an important feature in neurodegenerative disorders. HDAC6 inhibitory activity leads to increased acetylated HSP90 levels, thus prompting dissociation of HSP90-HDAC6 complex, and triggering ubiquitination of phosphorylated tau proteins (Figure 4).51


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Figure 4. Interaction of HDAC6 with ubiquitin and tau protein. 3.6 HDAC6 and TGF-β. Epithelial-mesenchymal transition (EMT) encompasses loss of cell-cell junctions and polarization of cell-surface molecules of epithelial cells, wherein they acquire the typical features of mesenchymal cells, including metastasis formation.52 Transforming growth factor-β (TGF-β) family and its members are well-known to be potent inducers of EMT. In particular, abnormal expression of TGF-β1 has been widely documented in the tumor microenvironment and fibrotic lesions.53 Studies performed on human lung epithelial cells demonstrated that TGF-β1 causes HDAC6-associated deacetylation of α-tubulin accompanied by increased cell motility and expression of EMT markers (Figure 5). Upregulation of HDAC6 by TGF-β1 occurs via post-translational mechanisms since TGF-β1 does not alter mRNA or protein levels of HDAC6. SMAD3 represents a vital element for TGF-β1-induced EMT through the canonical pathway and increasing data demonstrate that the microtubule structure controls SMAD activity.54 HDAC6 was found to regulate EMT through an interplay with SMAD3 signalling process, as demonstrated by the observation that TGF-β1-mediated phosphorylation of SMAD3



was impaired in the presence of HDAC6-specific siRNA. Also, TGF-β1-dependent nuclear accumulation of SMAD3 was found to be regulated by HDAC6.55

Figure 5. HDAC6-dependent and independent activation of EMT by TGF-β1. 3.7 HDAC6 and peroxiredoxins. Peroxiredoxin I (Prx I) and II (Prx II) are proteins involved in redox regulation; they are HDAC6 substrates and their acetylated forms are accumulated in the cells lacking HDAC6 activity.56 They belong to the Prx protein family, and specifically they represent the 2-cysteine members of the family, behaving as antioxidants at low H2O2 concentrations,57,

58

while at high concentrations of H2O2, oxidation of the cysteine residue to

sulphonic acid occurs, leading to the formation of high molecular-mass protein complexes. It has been observed that the acetylated Prx protein more actively reduces H2O2 than the non-acetylated form. Therefore, increased levels of acetylated Prx upon HDAC6 inhibition may result in a


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valuable enhancement of the antioxidant activity. In this regard, several HDAC inhibitors such as trichostatin A (TSA, 5, Figure 6 and Table 2), SAHA and sodium butyrate demonstrated efficacy in animal models of Huntington’s disease (HD)59 and amyotrophic lateral sclerosis.60 However, detailed mechanistic reasons for this beneficial effect are not clearly understood yet. Considering the unique place occupied by HDAC6 in the therapeutic spectrum, owing to its structural and functional features, several research groups have developed novel, potent and selective HDAC6 inhibitors for various disease profiles.61-64 In this aspect, the recently reported crystal structures of HDAC6 have helped advance newer drug design and discovery strategies towards partially selective HDAC6 inhibitors65 to be used in cancer therapy (e.g. ACY-1215, ricolinostat, 6, Figure 6 and Table 2).66, 67 Moreover, there has also been a recent surge in reports regarding the therapeutic application of novel selective HDAC6 inhibitors in non-cancerous conditions. This perspective article highlights the new guise of HDAC6 inhibitors as a therapeutic option for the treatment of diverse rare diseases.

4. HDAC6 inhibitors in rare disorders related to impaired axonal transport. As mentioned above, HDAC6 catalyzes the deacetylation of substrates with cytoplasmic localization, such as HSP90, α-tubulin, and cortactin.35, 68-70 Therefore, HDAC6 deficiency in cells leads to increased acetylation within the entire microtubule network.71 A number of evidences demonstrate that in neuronal cells the motor-based cellular trafficking is strongly dependent on the tubulin acetylation status, through the control of microtubule association with dynein and kinesin, which prompts amplified motor processivity and secretory vesicle flux.72, 73

These data also support the role of HDAC6 as a key regulator of endocytic cargo transport, due



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to its crucial role in modulating α-tubulin acetylation status.74 More in detail, the tubulin binding ability and motility of kinesin-1 (a motor protein responsible for the transport of cargos along microtubules) is regulated by the acetylation of α-tubulin at lysine residue 40. Inhibition of HDAC6 mediates, through augmented acetylated α-tubulin levels, the transport and accumulation to neurites of a kinesin-1 cargo protein, namely the JNK-interacting protein 1 (JIP1).73,

75

Moreover, it was demonstrated that pharmacological inhibition of HDAC6 with the pan-inhibitor TSA, beyond promoting in vivo dynein and kinesin-1 binding to microtubules, was able to boost the intracellular shuttling of the brain-derived neurotrophic factor (BDNF), another kinesin-1 cargo protein.72 On the contrary, defective α-tubulin acetylation reduces both recruitment of motor complexes and BDNF shuttling.72 Taken together, these evidences provide a clear-cut picture of the key role of HDAC6 in the control of microtubule-mediated intracellular trafficking, through its tubulin deacetylase activity.75 Therefore, HDAC6 is increasingly emerging as a valuable therapeutic target for the treatment of neurodegenerative diseases, including rare disorders.76 Already more than a decade ago, the neuroprotective effects associated to HDAC inhibitors (i.e., TSA and SAHA) have been highlighted in cell-based models of HD, in which they corrected the mutant huntingtin-induced deficits in vesicular BDNF transport.72,

77

More recently,

neuroprotective effects upon genetic ablation of HDAC6 have been demonstrated in vivo.78 Several pieces of evidence have also shown the protective effect of HDAC6 inhibition by enhancement of the axonal transportation of neurotrophic factors in in vivo models of brain disorders characterized by cognitive deficits.79, 80 This impaired BDNF transport has been related to rare disorders, such as RTT.81 Moreover, defective mitochondrial transportation and degradation of protein aggregates are abnormalities encountered in neurodegenerative disorders which are related to both catalytic and non-catalytic functions of HDAC680, such as CMT disease.82


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Accordingly, it has been demonstrated that HDAC6 silencing improved associative and spatial memory formation, which are impaired due to defective mitochondrial trafficking associated to neurodegenerative disorders.75, 83

4.1. HDAC6 and Rett syndrome (RTT) RTT is a rare neurodevelopmental disorder, mainly ascribable to the loss of functional mutations in the X-linked MeCP2 gene.84, 85 RTT patients are characterized by severe neurological, motor and behavioural defects. RTT clinical course entails the increasing impairment of intellectual functioning and motor skills, and defective head growth. Also, between 6 and 18 months after birth, RTT patients display stereotypic hand movements.86-88 MeCP2 interacts with DNA in the context of CpG islands behaving as a transcriptional repressor.8991

A series of evidences correlate the MeCP2 abnormalities with defective BDNF trafficking and

microtubule dynamics, thus highlighting a crucial role of HDAC6 in RTT neurobiology and its potential as a valuable disease-modifying target.92 MeCP2 controls Bdnf expression through its binding to the Bdnf promoter.93-95 Accordingly, reduced BDNF mRNA and protein levels were detected in MeCP2 deficient mice and patients affected by RTT.96-99 Moreover, Bdnf mutant mice and MeCP2 knockout mice exhibited comparable RTT phenotypes. Interestingly, Bdnf overexpression corrected several functional defects encountered in MeCP2 mutants, thus allowing to increase their lifespan.84,

96

Taken

together, these findings strongly suggest a key role for BDNF in neurological dysfunctions in RTT. As mentioned above, the acetylation levels of microtubules regulate the efficacy of a series of key processes in neuronal cells, namely differentiation and migration, and trafficking of mRNA,



mitochondria and BDNF-containing vesicles.72, 100 Furthermore, HDAC6 inhibition increased fast axonal transport of BDNF and mitochondria in both the anterograde and retrograde directions,72, 73, 101

thus enhancing synaptic activity.

MeCP2-mutated fibroblasts show a significant impairment in microtubule dynamics, displaying higher sensitivity towards cold-induced microtubule depolymerisation with the concomitant defective ability to recover.102 Similarly, defective microtubule dynamics were detected in astrocytes from RTT mouse models.103 Most importantly, impaired microtubule-mediated transportation of BDNF vesicles was described in a rodent model of RTT.104 The essential role of these processes for synaptic function and plasticity can help shed light on the pathways contributing to the neuropathology of RTT.92 In the context of RTT and HDAC6, reduced acetylated α-tubulin levels in cells from two male patients displaying different MeCP2 mutations when compared to control fibroblasts were recently demonstrated102. HDAC6 but not SIRT2 was shown to be a RTT biomarker in MeCP2 cells and in a MeCP2T158A RTT murine model. Tubastatin A (Tub A, 7, Figure 6 and Table 2) is a partially selective HDAC6 inhibitor able to cross the blood-brain barrier.77, 105 It was also shown to stabilize the microtubule network by counteracting depolymerization induced by either cold or nocodazole.106 Moreover, TubA induces a strong increase of acetylated tubulin levels, consequently ameliorating microtubule instability in RTT patient fibroblasts.92 These observations strongly suggest that augmented levels of acetylated tubulin in MeCP2/ MeCP2-deficient cells could be a direct effect of the HDAC6 overexpression and that treatment with a HDAC6 inhibitor could potentially lead to improved efficacy of molecular motors and motor-based trafficking of BDNF-containing vesicles, eventually enhancing synaptic activity.92, 107 TubA also stabilized the


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microtubule network of MeCP2-mutated fibroblasts, effectively counteracting nocodazoleinduced depolymerization, thus restoring polymerization levels to that of control fibroblasts.92 Moreover, employing a novel method to detect BDNF secretion, using surface immunostaining of BDNF tagged with a yellow fluorescent protein (BDNG-YFP) in live neurons after neuronal depolarization,108 the defective BDNF release was confirmed in hippocampal neuronal cells derived from MeCP2-null mice.99 Since increased acetylated α-tubulin levels improve microtubule stability, and BDNF vesicles are shuttled through the microtubule network,77 the use of selective HDAC6 inhibitors could represent an innovative targeted strategy for ameliorating cellular and synaptic defects in RTT.109 Despite the growing insights on MeCP2 function and the evidence of the efficacy of HDAC6 inhibitors in the frame of RTT in cells and animal models, the pathomechanisms underlying RTT are still not fully understood. Most importantly, animal models may not entirely recapitulate the features of the human disease, thus rendering difficult the identification of effective diseasemodifying therapies.110 A new frontier in this field was represented by the genetic reprogramming approach which allowed the development of induced Pluripotent Stem Cells (iPSCs) from human fibroblasts.111 Neurons deriving from iPSCs are mostly superimposable to neurons from human fetal brain112-114 and display a significant transcriptome resemblance to fetal neurons.115 It was demonstrated that iPSCs can be established from MeCP2-mutated patients,116,

117

thus

supporting the validity of iPSCs technology for recapitulating RTT features in vitro.118, 119 MeCP2iPSCs neurons show a reduced number of synapses, impaired spine density, and defective calcium signalling when compared to controls. These phenotypic alterations are completely superimposable to those observed in RTT mice and post-mortem brain tissues derived from patients’.118



A transcriptome profiling of MeCP2-iPSCs neurons was performed in order to unveil the specific signature of RTT neurons responsible for the observed phenotypic changes. This analysis highlighted a significant GABAergic neurons impairment in RTT pathogenesis. 81 Most importantly, RNA-sequencing results highlighted an abnormal expression of the HDAC6 gene, further confirmed by Western blot analysis evidencing a relevant reduction of acetylated αtubulin levels in MeCP2-iPSCs neurons, likely as the direct result of the increased HDAC6 activity. In support of the hypothesized connection between HDAC6 overexpression and decreased acetylated tubulin levels, treatment of MeCP2-iPSCs neurons with a new class of selective HDAC6 inhibitors (general structure 8, Figure 6 and Table 2), effectively increased the levels of acetylated α-tubulin, as determined by Western blot analysis on mutated and control neurons.81 Consequently, scientists very recently proposed the repositioning of ricolinostat, currently in clinical trials for the therapy of multiple myeloma in association with proteasome inhibitors, such as bortezomib.120,

121

Accordingly, the treatment with ricolinostat led to increased levels of

acetylated α-tubulin, thus further supporting the close connection between HDAC6 deacetylase activity and the status of the entire microtubule network. In conclusion, a significant number of experimental findings point out that microtubule-based trafficking is strongly related to the acetylation status of tubulin and impairments in its fine-tuning are strongly implied in defective bi-directional trafficking of BDNF, vesicle transportation, neuronal cell migration and axon elongation. The impact of HDAC6 expression on these pathways, through its unique tubulin deacetylase activity, strongly supports its role as a potential therapeutic option to counteract cellular and synaptic defects in RTT.


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4.2. HDAC6 and Charcot-Marie-Tooth disease (CMT) CMT can be classified as a heterogeneous group of hereditary motor and sensory peripheral neuropathies (HMN) affecting 17–40 per 100,000 individuals. Nearly 40 genes have been related with CMT pathogenesis.122, 123 The vast majority of affected individuals exhibit progressive muscle weakness and atrophy. At the later CMT stages, muscles of hands and forearms are involved. In 90% of cases CMT shows an autosomal dominant pattern of inheritance.123, 124 Based on electrophysiological features, CMT can be clustered into three main groups: type 1 CMT (CMT1), mainly characterized by demyelination, type 2 CMT (CMT2), mainly featuring axonal loss, and intermediate forms displaying both axonal loss and demyelination.125 Although CMT displays genetic and clinical heterogeneity a number of studies highlighted common features. Therefore, despite the variety of primary causes which may be involved, the mechanisms underlying the disease seem to be mostly related to abnormalities in the cytoskeleton of peripheral axons and to defective axonal transport.126 Currently, the few drugs available for the treatment of CMT act by halting/slowing down disease symptoms, while a disease-modifying approach is still missing. In 2010, Yamauchi and collaborators showed that valproic acid (VPA, 9, Figure 6 and Table 2), an antiepileptic drug also known for its activity as a pan-HDAC inhibitor, was able to restore neurite formation events in the CMT2B mutant Rab7. However, the authors correlated this protective effect to the modulation of the JNK signalling pathway, rather than to the inhibition of HDAC activity.127 One year later, Van Den Bosch and co-workers characterized two mutant murine models expressing mutant small heat-shock protein B1 (HSPB1), a 27 kDa small HSP, accurately



recapitulating human CMT2 and distal HMN.82 The authors investigated the outcome of mutant HSPB1 on mitochondria axonal transport in dorsal root ganglion (DRG) neurons isolated from transgenic mice. The authors were able to measure the number of mitochondria and to distinguish bi-directionally moving mitochondria from the motionless ones. DRG neurons of HSPB1S135F mutant showed a reduced number of mitochondria compared to their wild-type littermates, accompanied by significantly reduced moving mitochondria in their neurites.82 In agreement with impaired mitochondrial transport, they also registered a significant reduction of acetylated αtubulin levels in the mutated mice peripheral nerves. Since HDAC6 has a key role in controlling the axonal transport of mitochondria in cultured hippocampal neurons101 and in order to understand the mechanisms underlying CMT2 induction by HSPB1 mutants, the authors incubated DRG neurons from HSPB1S135F mutant with diverse HDAC6 inhibitors. In particular, the pan-inhibitor TSA and the selective HDAC6 inhibitors TubA and tubacin (10, Figure 6 and Table 2) were investigated. The three inhibitors were able to nicely increase the total number of mitochondria; interestingly, TubA and tubacin were more effective in restoring the number of moving mitochondria, thus highlighting the key role of HDAC6 isoform in the microtubule network impairment associated to CMT disease.82 As the following advancement, evaluation of the best-performing inhibitors was translated to animal models. Symptomatic treatment of mutant mice with TSA and TubA for 21 days provided a robust increase of acetylated α-tubulin levels in peripheral nerves and led to significantly improved motor performance. Taken together, these results strongly pointed out to HDAC6 as a novel potential therapeutic option for an effective disease-modifying approach for CMT and other peripheral nerve disorders.82, 83, 125, 128


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Recently, a novel HDAC6 inhibitor characterized by improved selectivity (11, Figure 7 and Table 2) was evaluated in cell-based settings, namely the Neuro-2a (N2a) cells, in which the effects on the acetylation levels of histone H3 and α-tubulin (as markers of inhibition of class I HDACs and HDAC6, respectively) were evaluated.129 Compound 11 effectively increased the levels of acetylated α-tubulin, while not altering the acetylation levels of histone H3. The compound was further investigated in vitro in cultured DRG neurons from a mouse model of CMT2 induced by mutant HSPB1. In this model, compound 11 was able to improve mitochondrial transport, thus correcting the CMT-associated mitochondrial axonal transport more effectively than other unselective HDAC6 inhibitors. Another recent study was engaged on three potent HDAC6 inhibitors under preclinical evaluation or in clinical trials, namely ACY738 (12, Figure 7 and Table 2), ACY775 (13, Figure 7 and Table 2) and ricolinostat.130 The advantage of these three molecules is their drug-like profile, suitable for the transition into clinical use as a potential targeted therapy for CMT disease. The more selective HDAC6 inhibitors 12 and 13 were investigated in cultured DRG neurons from HSPB1S135F mice and from adult non-transgenic mice (NTG) for their ability to improve mitochondrial axonal transport. Immunofluorescence analysis highlighted a relevant increase of acetylated α-tubulin induced by treatment with either 12 and 13. DRG neurons cultured from symptomatic HSPB1S135F were treated with the two inhibitors (2.5 μM dose) to assess their ability to counteract CMT-related mitochondrial axonal transport defects. Both compounds were able to significantly increase the motility and the total number of mitochondria in comparison with non-treated DRG neurons. In vivo transition of these molecules in the HSPB1S135F-induced CMT2 mouse model gave the opportunity to evaluate their effects on motor and sensory deficits. After daily intraperitoneal administration of 12 and 13 (3 mg/kg), or ricolinostat (30 mg/kg) for 21 days, motor and sensory



nerves were submitted to nerve-conduction studies, which showed that all tested inhibitors were able to increase the action potential amplitudes. In order to visualize reinnervation of the neuromuscular junctions, the mice were sacrificed, and the gastrocnemius muscle was dissected. Quantification of the number of double-labelled neuromuscular junctions highlighted significant reinnervation following treatment with the three HDAC6 inhibitors, thus confirming the potential of the compounds to improve motor and sensory deficits in a CMT2 animal model.130 Due to the phenotypic similarities encountered in CMT individuals and those with vincristineinduced peripheral neuropathies (VIPN), it was also recently assumed that vincristine could cause axonal deficits by altering microtubule dynamics. Therefore, HDAC6 inhibitors were investigated as a potential treatment of VIPN. Beyond the reduced proliferation of lymphoblastic T cells associated to HDAC6 inhibition, the significant therapeutic effects registered in VIPN rodent models were related to the augmented acetylated α-tubulin levels, responsible of restoring the axonal transport.131 Very recently, Mo and co-workers added a further advance to the study of the pathomechanisms in CMT disorder, analyzing the interaction of Glycyl-tRNA synthetase (GlyRS) and HDAC6.132 GlyRS was the first member identified, among the dominant mutations associated with CMT2D. However, it was demonstrated that CMT2D phenotype could not be recapitulated by loss of wildtype protein function, but it could rather be associated with abnormal activities of mutant GlyRSCMT2D. The authors identified HDAC6 as an intracellular element abnormally interacting with GlyRSCMT2D. This abnormal interaction fuels HDAC6 catalytic activity, thus negatively impacting on α-tubulin acetylation levels. A HDAC6 inhibitor (administered i.p. for 2 weeks at 50 mg/kg) was able to restore axonal transportation and to rescue motor functions in CMT2D mice,


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thus further highlighting HDAC6 role as a pathogenic factor and therapeutic target in CMT disease. Almost in parallel, Van Den Bosch and collaborators provided proof for the interaction of GlyRS and HDAC6 through a coimmunoprecipitation assay in N2a cells.133 In line with the previous evidence, GlyRS-HDAC6 interaction was disrupted in cells incubated with TubA prior to coimmunoprecipitation. Also, an increased level of acetylated α-tubulin was detected in cell lysate in response to treatment with TubA. Moreover, the study demonstrated that acetylation tissue levels from GarsC201R/ + mice were significantly decreased relative to sciatic nerve tissues. Also, mitochondrial transport was impaired in DRG cultures from GarsC201R/

+

mice. TubA was

intraperitoneally administered to GarsC201R/ + mice; subsequently, motor behaviour and parameters related to nerve conduction were assessed, thus demonstrating the beneficial effects of drug treatment in mutant mice. Analogously to what described for RTT, ex vivo cellular models of disease based on patientderived iPSCs provide powerful approaches for the dissection of the molecular underpinnings of CMT disease biology. In a study conducted by Kim and collaborators with iPSCs from CMT disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) carrying heat shock 27 kDa protein 1 (HSPB1).134 The mutant cell lines showed decreased levels of acetylated αtubulin and relevant deficits in the axonal movement of mitochondria. Two novel HDAC6 inhibitors were employed in the study. Both the assayed inhibitors were able to increase the acetylation level of α-tubulin and to rescue the impairment of axonal mitochondrial trafficking.



5. HDAC6 and inherited retinal diseases (IRDs) IRDs are a group of pathologies featuring a progressive retinal degeneration.135 In most cases, mutations of genes expressed either in the photoreceptor cells (rod or cones) or in retinal pigment epithelium are involved.136 These mutations cause alterations in the correct functioning of photoreceptors leading to slow and progressive degeneration of the retina with consequent blindness. In Retinitis Pigmentosa (RP), mutations mainly occur at rod photoreceptors, followed by secondary cone photoreceptors damage. In diseases such as Best’s and Stargardt’s disease, dystrophies of the cone photoreceptors are involved. Activation of HDACs in animal models of IRDs was documented.137-139 In pre-clinical rodent models of both rod and cone photoreceptors degeneration, class I/II HDAC inhibition was demonstrated to have protective effect against photoreceptors degeneration, even though no data were initially presented regarding specific HDAC sub-type involvement.138, 140 Among the RP murine models available, the retinal degeneration 1-(rd1) mouse model, the first mouse model of retinal degeneration initially referred to as the rodless mouse, carries a mutation in the gene encoding for the rod photoreceptor cGMP phosphodiesterase-6 (pde6), causing an increase in cGMP levels and oxidative stress.141-143 An increased HDAC activity in rd1 mutated photoreceptors, with resulting acetylation/deacetylation imbalance could be involved in their degeneration.

139

Inhibition of HDAC activity by TSA or scriptaid (14, Figure 7 and Table 2)

resulted in a neuroprotective effect to rd1 mutated photoreceptors and retinal explant cultures. Using the rd10 mouse model in which rod cell death is ascribable to a mutation in the rod-specific Pde6b gene, Trifunovic et al.144 demonstrated that rd10 cultures treated with 10 M SAHA every other day for 14 days showed a five-fold increase of surviving rd10 mutant photoreceptors. The


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cone-photoreceptor-function-loss-1 (cpfl1) mouse model presents a mutation in the cone-specific Pde6c gene causing an early-onset (post-natal day 14 – PN14) and fast (peak of cell death at PN24) loss of cones. Using this model, Trifunovic et al. demonstrated that both at PN14 and at PN24 cpfl1 cone photoreceptors show increased HDAC activity.137, 144, 145 Treatment of cultured cpfl1 retina cells explanted at PN14 with TSA (10 nM) every second day until PN24 resulted in an increase of surviving cones with respect to untreated control.145 Also in vivo intravitreal injection of TSA was shown to protect degenerating cones. Since cpfl1 retina displays an aberrant cone positioning, treatment with TSA improved the altered cone migration.145 In 2011, Clemson et al.146 reported a study on the off-label use of VPA,147 for the treatment of RP. Enhanced visual field and acuity and deferred vision loss were reported, posing the basis for subsequent approval of a randomized, placebo-controlled trial for the treatment of autosomal dominant RP. However, the original study was subject to criticism due to problems related to experimental design and results analysis.146, 148-150 Another study with longer patient follow up, involving patients with several retinal dystrophies showed no benefits of VPA treatment and highlighted adverse effects of the drug.151 Moreover, VPA was demonstrated to have a beneficial effect in an in vitro model characterized by P23H mutation of rhodopsin, while it had a detrimental effect on the degeneration of the retina in the T17M model of RP, this latter effect seemingly due to inhibition of HDAC.152 The role of HDAC6 in retinal degeneration was specifically investigated by Leyk et al.153 who demonstrated that mouse-derived cone-like 661W cells do express HDAC6 and that the enzyme is active in that model since treatment with specific inhibitors led to -tubulin hyperacetylation. Inhibition of HDAC6 protected the cells from oxidative stress induced by H2O2. In vivo studies were conducted on the dyeucd6 zebrafish model, displaying defective visual behavior and retinal morphology. HDAC6 inhibitors induced an increase in eye saccades on treated dye larvae and



enhanced locomotor activity in response to light as assessed in the visual-motor response assay. Investigation of the molecular basis of the observed protection highlighted a significant upregulation of HSP25 and HSP70 in 661W cells upon treatment. However, other experiments highlighted that this effect alone could not fully explain the protective effect of TubA. Further investigations were performed on Prx1, an enzyme involved in the detoxification of H2O2, which is overoxidized and inactivated under oxidative stress conditions and a known substrate of HDAC6. HDAC6 inhibitor pretreatment reduced the levels of PrxSO2/3 (the inactivated form of Prx1) with respect to H2O2-treated controls, so its effect could be mediated by regulation of Prx1 activity. Actually, it is known that Prx1 reducing activity is also controlled by HDAC6-mediated post-translational acetylation.25 Consequently, HDAC6 inhibition has a significant protective role from oxidative stress-induced injuries and this is particularly important for photoreceptors and retinal cells which are exposed to high concentrations of reactive oxygen species (ROS).

6. HDAC6 and Idiopathic Pulmonary Fibrosis (IPF) IPF is a chronic, progressive interstitial lung disease (ILD) with indefinite etiology and characterized with a poor prognosis. Pathological hallmarks of IPF are fibroblast activation which implies aberrant proliferation, myofibroblast transformation, tissue invasiveness, and extracellular matrix production.154 Epidemiological data indicate that the incidence of IPF in Europe and North America ranges between 2.8 and 18 cases per 100,000 people per year with possibly lower numbers in Asia and South America (from 0.5 to 4.2 cases per 100,000 individuals per year) with


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an average life expectancy from diagnosis of 2–4 years (the median age at diagnosis is usually around 65 years).155 Despite the recent introduction in the therapy of nintedanib156 (a tyrosine kinase inhibitor), and pirfenidone157 (anti-fibrotic and anti-inflammatory agent acting, with a not fully elucidated mechanism of action yet) the IPF medical need is high. Hence research is ongoing in the quest for new therapeutic options and to glean novel insights towards innovative and druggable targets for the therapy of this life-threatening disease. The mechanisms relevant for IPF pathogenesis have not fully elucidated, however, may offer a glimpse in the therapeutic targeting of the disease. In the context of epigenetic targets, the involvement of HDACs in IPF has been proved by a limited number of seminal studies. Deacetylation of non-histone proteins controls several key cellular processes (cell signalling, motility, survival, protein degradation, and inflammation) and, as pointed out by some recent research reports, also plays a pivotal role in fibrotic degeneration.158 Fibrosis results from an overgrowth of various tissues, an increased number of myofibroblasts and is accompanied by an abnormal deposition of extracellular matrix components. EMT is a key process in IPF. It is known that in lung fibrogenesis a relevant number of myofibroblasts can generate via EMT, starting from the resident epithelial cells. EMT is highly stimulated by TGFβ1. In 2008 Shan and colleagues explored the molecular pathways underlying TGF-β1-induced EMT, and proposed HDAC6 as a novel regulating factor for TGF-β1-induced EMT.159 Accordingly, HDAC6 inhibitor tubacin decreased the expression and generation of TGF-β1induced EMT markers (namely epithelial and mesenchymal peptides, and stress fibers) to the same extent as that produced by small interfering RNA. An cross-talk between HDAC6 and SMAD3 was also evidenced in TGF-β1-induced EMT. Shortly after, Wang and coworkers confirmed the



therapeutic benefits associated to use of HDAC inhibitors in IPF by applying the HDAC paninhibitor SAHA.160 This latter compound impaired TGF-β1-induced fibroblast-myofibroblast differentiation and inhibited fibroblast proliferation. Later, a crucial role of HDAC4 in TGF-β1stimulated trans-differentiation of lung fibroblasts via a mechanism regulating Akt phosphorylation was demonstrated. However, in this mechanism, possible cooperation of HDAC4 with other HDACs could not be ruled out.161 As opposed to the role of TGF-β1, that acts as a profibrotic mediator, the prostaglandin E2 (PGE2) acts as antifibrotic mediator in IPF. PGE2 is produced via the cyclooxygenase (COX) pathway. Of the two COX isoforms (COX-1 and COX-2), COX-2 is inducible by inflammatory stimuli including TGF-β1. Interestingly, Coward and colleagues found lower COX-2 mRNA levels in fibroblasts from IPF patients and described an epigenetic aberration leading to deregulated gene expression in IPF.162 They demonstrated that reduced COX-2 expression in IPF is due to histone hypoacetylation. Accordingly, when fibroblasts from IPF patients underwent treatment with SAHA or panobinostat, histone H3 and H4 acetylation levels were increased, thus ultimately restoring cytokine-induced COX-2 mRNA and protein expression. In 2010 the same team, by capitalizing the data from the previous study, completed a new investigation focusing on angiogenesis in IPF.163 Their working hypothesis was that an aberrant angiogenesis supporting tissue remodelling might contribute to IPF pathogenesis in similar fashion to that observed in tumorigenesis. In particular, the authors observed that the angiostatic chemokine gamma interferon (IFN-)-inducible protein of 10 kDa (IP-10), that acts as an antifibrotic by modulating fibroblast migration and proliferation, is repressed in IPF fibroblasts. They observed that interplay between histone deacetylation and hypermethylation is accountable for the decreased expression of IP-10. To confirm this hypothesis, treatment with the pan-HDAC inhibitor panobinostat, or with a G9a


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inhibitor, restored IP-10 mRNA and protein expression in fibroblasts from IPF patients. However, the detailed mechanisms underlying restoration of IP-10 gene expression induced by HDAC inhibitors require further investigation. More recently, SAHA has been shown to induce apoptosis of IPF myofibroblasts through upregulation of the pro-apoptotic gene Bak and parallel downregulation of the antiapoptotic gene Bcl-xL.164 Since in fibroproliferative diseases, such as IPF, are characterized by resistance to apoptosis, the epigenetic pathways accountable for the reduced expression of Fas, a cell surface death receptor decreased in IPF, were further explored.165 Reduced Fas expression and resistance to Fas-mediated apoptosis were registered in fibroblasts deriving from bleomycin-treated mice. Also, reduced histone acetylation and augmented histone H3 lysine 9 trimethylation (H3K9Me3) levels were detected in the Fas promoter in the same cell type. These events were related to HDAC2 and HDAC4 overexpression. Accordingly, treatment with HDAC pan-inhibitors enhanced Fas expression and reestablished susceptibility to Fas-mediated apoptosis. Further insights on the role of HDACs in IPF were recently provided. Korfei demonstrated an abnormal overexpression of HDAC enzymes in basal cells of IPF lungs and how it contributes to the bronchodilation process.166 These studies led to the conclusion that the enhanced activity of HDACs mediates the formation and apoptosis resistance of IPF fibroblasts.167 This pioneering study also provided evidence that panobinostat was able to downregulate the biosynthesis of collagen I and antiapoptotic genes in IPF fibroblasts, thus correcting their profibrotic and ‘malignant’ phenotype. Although no hints on differential involvement of HDAC isoforms in IPF were provided, it was ascertained that pan-HDAC inhibition may represent a valuable treatment option for IPF patients. In fact, by a comparative analysis with pirfenidone, they observed a higher



efficacy of panobinostat in reducing the profibrotic phenotypes of the IPF fibroblasts while inducing cell cycle arrest and apoptosis. On these premises, a series of pan-HDAC inhibitors were then tested on a variety of cell and animal models by other research groups. Recently, Davies tested Spiruchostatin A (15, Figure 7 and Table 2) a cysteine-containing depsipeptide binding HDAC6, in primary fibroblasts from lung biopsy explants of IPF patients.168 The compound was able to decrease the aberrant proliferation of IPF already in the nanomolar range. Notably, the effective dose of 15 was significantly lower than that of either inhibitor TSA161 or SAHA160 as seen from previous studies, with a markedly longer duration of action. The antifibrotic efficacy of romidepsin was investigated in vitro and in vivo.169 The efficacy of the tested compound in inhibiting fibroblast proliferation was established with no adverse effects on alveolar type II cells, where HDAC6 is predominantly overexpressed in IPF.170 When tested in vivo, in a rodent bleomycin model of IPF, romidepsin inhibited fibrosis. Notably, this effect was paralleled by suppression of lysyl oxidase (LOX) expression, taken as a companion biomarker of the activity of the inhibitor. LOX was identified as a valuable biomarker as its activity has strong connections with organ fibrosis. In fact, LOX is induced by the profibrotic factor TGF-β1, which is in turn strongly related to HDAC6-mediated α-tubulin deacetylation responsible for the increase of EMT markers in lung epithelial cells. Therefore, romidepsin may counteract IPF fibrosis targeting multiple pathways. More recently it was proved that HDAC inhibitors modulate exchange factor found in platelets, leukemic, and neuronal tissues (XPLN) expression and affects secreted protein acidic and rich in cysteine (SPARC) expression in human lung fibroblasts.171 SPARC contributes to the deposition


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and turnover of the extracellular matrix, controlled by TGF-β1 by activation of mammalian target of rapamycin complex 2 (mTORC2) which is modulated by XPLN. Fibroblasts were treated with TGF-β1 and HDAC inhibitors entinostat (16, Figure 7 and Table 2) or SAHA, to evaluate their influence on XPLN expression and demonstrated that: i) the HDAC inhibitors dose-dependently up-regulated XPLN mRNA, ii) TGF-β1-induced SPARC expression was counteracted upon exposure to entinostat. By seminal studies, the role of XPLN in the regulation of SPARC was demonstrated indicating that the up-regulation of XPLN induced by HDAC inhibitors may represent a useful therapeutic strategy for IPF patients. Once the role of HDACs was established in IPF, the involvement of specific HDAC isoforms was further and widely investigated in the very recent years. Saito and colleagues addressed the relevance of selectively targeting HDAC6 for the treatment of IPF.172 Interestingly, HDAC6 expression in lung tissue homogenates derived from patients affected or not from IPF was examined and an aberrant HDAC6 expression in IPF lungs was observed. In fact, inhibition of HDAC6 can alter the activity and fate of several proteins and implication of HDAC6 in TGF-β1-induced EMT and Akt signalling pathways (in fact TGF-β1 prompts myofibroblast differentiation by activation of SMAD3 and Akt signalling pathways) was confirmed. In these studies, HDAC6 inhibitors ricolinostat, TubA , tubacin and 17 (Figure 7 and Table 2) were tested. TubA was the only inhibitor that, when tested in the bleomycin murine model of IPF, significantly inhibited TGF-β1-mediated type-1 collagen expression, by reduction of Akt phosphorylation and regulation of downstream pathways such as HIF-1α-VEGF axis and autophagy (as noted by the decreased levels of the marker LC3B-II). These data indicate HDAC6 inhibitor-treated wild-type mice were protected against bleomycin-induced fibrosis, but HDAC6 knockout mice were not.



Further support to the role of acetylated α-tubulin in fibrotic degeneration as a marker of EMT and of HDAC6 as a key regulator of the EMT process in IPF has been provided.173 The effectiveness of tubacin was demonstrated in restoring acetylated α-tubulin levels and consequently blocking EMT. In particular, it was confirmed that: i) acetylated α-tubulin levels are reduced during TGFβ-induced EMT; ii) loss of acetylated α-tubulin is a common feature in fibrotic cells during EMT; iii) high levels of acetylated α-tubulin block EMT; iv) TGF-β1prompts HDAC6 activity; v) increased expression of HDAC6 induces EMT in fibrotic cells. Beyond the traditional bleomycin mouse model for the in vivo validation of anti-fibrotic approaches in IPF, a very recent ex vivo model of human lung fibrogenesis has emerged. This model recapitulates TGF-β1-mediated events stimulating fibrosis in IPF. By preserving the multifaceted cell-cell and cell-matrix interplay found in human tissues, this model may be of extreme utility to accurately predict drug efficacy, thus accelerating drug discovery track in IPF, also with reference to HDAC inhibitors.174 In a very recent report, Lou and colleagues applied a drug repurposing approach to HDAC inhibitors to treat IPF and acute lung injury (ALI).175 Their working hypothesis was based on the evidence that both diseases are characterized by neutrophilic inflammation and that blockade of this event might represent a valuable treatment strategy for both ALI and IPF. In this context, among several chemotactic agents, leukotriene B4 (LTB4) represents the main player. In fact, it was noted that levels of this lipid mediator were persistently elevated in broncho-alveolar lavage fluid (BALF) in ALI models and in BALF from IPF patients. Furthermore, the levels of LTB4 well correlate with the degree of fibrotic degeneration in tissues. On this basis, the authors propose that blockade of LTB4 biosynthesis, by inhibiting leukotriene A4 hydrolase (LTA4H), would


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lower inflammation thus providing an alternative therapeutic approach in IPF or ALI. In their repurposing approach, the authors involved 18 HDAC pan-inhibitors. After enzymatic tests, thermofluor assays, and X-ray analysis they selected SAHA and its analogue 18 (Figure 7 and Table 2) as effective inhibitors of LTA4H. They observed that SAHA repressed neutrophils infiltration and LTB4 biosynthesis in murine models of both ALI and IPF. Compound 18 performed better than SAHA in the same models. Although they concluded that HDAC and LTA4H dual inhibitors display higher efficacy with respect to an inhibitor specifically targeting HDACs, we may argue that the engaged compounds hit too many targets for being used as useful pharmacological tools for the study of an innovative mechanism of action. In general, pan-HDAC inhibitors proved their effectiveness for the potential treatment of IPF. However, as isoform selectivity may imply a low attrition rate in the process of drug development, the identification of subtype-selective compounds may be preferred. In this context, the above literature reports support the predominant role of HDAC6 in EMT and validate this enzyme isoform as an innovative target for a ground-breaking targeted approach in IPF therapy.



O Me

O Me

N Me

N H

Me

OH

N

N

O

H N

N

OH

6, Ricolinostat, ACY1215

Me N

R

X

N

N H

6

O

5, Trichostatin A (TSA)

N

H N OH

O NH OH

O 7, Tubastatin A (TubA)

8 O

O HO

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HN

Me

Me 9, Valproic acid (VPA)

O

O 6 N H

O S

HO

OH

10, Tubacin

N

Ph

O Ph

Figure 6. Structures of HDAC inhibitors 5-10.


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N

H N

Me N

OH

O

12, ACY738

O

F

H N

N

NHOH 11

N

F H N

N H N

N

O

O Me

Me O

NH O N H

H N

OH

O

14, Scriptaid

13, ACY775

S

O

OH

O

S

N

Me O

OH

N H

NH2

N H

H N

O

N

O

O

O

16, Entinostat

15, Spiruchostatin A O

HN OH

O

O F 17, MC1568

N Me

Me

N H N Me

H N

6

OH

O

18, M344

Figure 7. Structures of HDAC inhibitors 11-18.



Table 2. IC50 values for HDAC inhibitors 1-18 on HDAC6 and other isoforms belonging to Class I.

Compound

IC50 HDAC6 (nM)

1, SAHA 2, Romidepsin 3, Belinostat 4, Panobinostat 5, Trichostatin A 6, Ricolinostat

7, Tubastatin A 8 9, Valproic acid 10, Tubacin 11 12, ACY738 13, ACY775 14, Scriptaid 15, Spiruchostatin A 16, Entinostat 17, MC1568 18, M344

IC50 Class I HDACs (nM) HDAC1 = 33 HDAC2 = 96 33 HDAC3 = 20 HDAC8 = 540 HDAC1 = 1.6 790 HDAC2 = 3.9 82 HDAC1 = < 15 < 15 HDAC3 = < 15 HDAC8 = 550 8.6 HDAC1 = 6 HDAC1 = 58 HDAC2 = 48 4.7 HDAC3 = 51 HDAC8 = 100 HDAC1 = 16,400 HDAC2 = >30,000 15 HDAC3 = >30,000 HDAC8 = 854 56 HDAC1 = 2,570 HDAC1 = 40,000 HDAC1 = 1,400 HDAC2 = 6,270 4.0 HDAC3 = 1,270 HDAC8 = 1,270 3.0 HDAC1 = 4,320 1.7 HDAC1 = 94 7.5 HDAC1 = 2,123 HDAC1 = 600 HDAC3 = 600 HDAC8 = 1,000 1600 HDAC1 = 3.3 HDAC1 = 510 HDAC3 = 1,700 HDAC = 100 nM (maize HD1-A ) HDAC = 100 nM (maize HD-2)


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7. Perspective Many research efforts in the past two decades have been devoted to the identification of relevant epigenetic therapeutic targets in cancer and to the design of epigenetic therapies effective in tackling crucial oncological pathways. More recently, growing evidence indicates that epigenetic pathways are pertinent to many other non-oncological diseases, particularly rare diseases. Rare diseases represent an unmet medical need due to their low prevalence which in turn leads to poor interest from both academia and industry in the development of specific therapies. Moreover, the patients diagnosed with rare diseases encounter unique challenges that initiate with lengthy diagnosis times, as many health care experts have limited experience in identifying rare diseases and may miss the initial symptoms. In addition, the scarce availability of suitable disease models has dissuaded focused efforts to address this imminent challenge. The past few years have represented a phase of hope for patients, with increased scientific awareness towards rare disease therapeutics. Accordingly, researchers in academia and pharma are actively collaborating to leverage new technologies and scientific knowledge on these diseases for developing therapies. Such efforts have received impetus from the successful completion of the Human Genome Project in 2003 and from the progress in proteomics and next-generation sequencing. However, the identified targets from such techniques have yet to be validated as ‘druggable’. Unfortunately, this process usually narrows down the availability of suitable drug targets to an abysmally low number. Besides this, a continuous struggle in the quest for highly specific disease biomarkers for effective targeted therapy approaches has proved to be an existential issue. The recent progress in (epi)genomics and proteomics have enabled to clearly unveil the molecular underpinnings responsible for disease initiation and progress. A number of rare involve



epigenetically regulated genes or other epigenetic components. While genetic mutations are infrequent, epigenetic modifications are very common. Therefore, the interplay between genetics and epigenetics plays a crucial role when considering the etiologic factors in some of these diseases. A very representative example is the Rett syndrome, which is caused by mutations of genes related to DNA methylation (namely mutations in the methyl-binding domain protein MeCP2). This genetic mutation is flanked by a series of epigenetic changes which strongly contribute to the disease phenotypic manifestations. In this context, HDAC enzymes have emerged as a central player in regulating pathophysiological mechanisms involved in specific rare diseases. HDAC6 stands out as a unique deacetylase isoform owing to its distinctive structure, domain organization and its cytoplasmic localization. The upregulation of HDAC6 levels has been confirmed in rare diseases, such as RTT and IPF among others. This evidence, coupled with the direct involvement of HDAC6 substrates found in disease models, strongly support the investigation of HDAC6 as an innovative and disease-modifying target in the treatment of such disorders. Both HDAC pan-inhibitors and HDAC6 partially selective modulators have been investigated in recent years in cell- and animal-based models of rare diseases, such as RTT, IPF, CMT and IRDs. We have herein described these approaches and showed that HDAC6 inhibition provided in many cases a robust proof-of-concept as a novel therapeutic option for these pathologies. Another recent advancement has been represented by the advent of novel and more reliable disease models (also with respect to rare diseases) employing the technology associated with iPSCs. These cells have a special resemblance with human tissues, thus providing highly reliable results and enhancing the successful translation of preclinical candidates into clinical studies. On the other hand, most of the approved HDAC inhibitors possess a low degree of selectivity for HDAC6 and


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are not useful to dissect the role of this specific isoform in these rare diseases and cannot be considered therapeutic options for treating these patients. The X-ray co-crystal structures with several HDAC6 inhibitors allowed more powerful and reliable structure-based design strategies, which led to conceive selective HDAC6 inhibitors. However, medicinal chemists are still called to invest more efforts in the fine-tuning of the drug discovery trajectory towards selective HDAC6 inhibitors, also endowed with a favourable druglike profile. In conclusion, it is conceivable that, in the new era of personalized medicine, epigenetic research involving HDAC(6) modulators, as well as modulators of other specific epigenetic targets, will lead scientists in the direction of developing individualized therapies in the area of rare diseases, in resemblance to what is being promised by personalized genomic-based medicine in other diseases, such as cancer.

ACKNOWLEDGEMENTS The Authors acknowledge MIUR Grant Dipartimento di Eccellenza 2018–2022 (l. 232/2016) to the Department of Biotechnology, Chemistry and Pharmacy, University of Siena and the Department of Pharmacy, University of Naples Federico II.

Abbreviation list ALI, acute lung injury; BALF, broncho-alveolar lavage fluid; BDNF, brain-derived neurotrophic factor; BDNG-YFP, BDNF tagged with a yellow fluorescent protein; CMT, Charcot-Marie Tooth disease; CNS, central nervous system; COX, cyclooxygenase; DNA, deoxyribonucleic acid;



dHMN2B, distal hereditary motor neuropathy 2B, DNMT, DNA methyltransferase; DRG, dorsal root ganglion; EGFR, epidermal growth factor receptor; EMT, epithelial-mesenchymal transition; FDA, Food and Drug Administration; GlyRS, Glycyl-tRNA synthetase; GR, glucocorticoid receptor; HD, Huntington’s disease; HDAC, histone deacetylase; HMN, hereditary motor neuropathies; HSF1, heat shock transcription factor 1; HSPB1, heat-shock protein B1; HSP90, heat shock protein 90; IFN-, gamma interferon; ILD, interstitial lung disease; IPF, idiopathic pulmonary fibrosis; iPSCs, Pluripotent Stem Cells; IRDs, inherited retinal disorders; JIP1, JNKinteracting protein 1; KATs, lysine acetyltransferases; LOX, lysyl oxidase; LTB4, leukotriene B4; MeCP2, X-linked methyl-CpG-binding protein 2; ncRNA, non-coding RNA; NES, nuclear export signal; NTG, non-transgenic mice; PLAP, phospholipase A2 inactivating protein; PTMs, posttranslational modifications; Prx, peroxiredoxin I; p97/VCP, p97/valosin containing protein; RP, retinitis pigmentosa; RTT, Rett syndrome; ROS, reactive oxygen species; SAHA, suberoylanilide hydroxamic acid; SIRT, sirtuin; TGF-β, transforming growth factor-β; TSA, Trichostatin A; Tub A, Tubastatin A; VEGF, vascular endothelial growth factor; VIPN, vincristine-induced peripheral neuropathies; VPA, valproic acid; UBPs ubiquitin-specific proteases; ZnF-UBP, ubiquitin Cterminus hydrolase-like zinc finger.

Corresponding Author G. Campiani, Email: [email protected].


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Biographical sketch Margherita Brindisi graduated cum laude from the University of Siena, where she also received her PhD in Pharmaceutical Sciences in 2008. In 2010-2011, she worked as a post-doctoral fellow at Purdue University on the development of aspartyl protease inhibitors under the supervision of Prof. Arun Ghosh. From 2012 to 2015 she was appointed as fixed-term Researcher at the Department of Biotechnology, Chemistry and Pharmacy, University of Siena, where she worked in the group of Prof. Giuseppe Campiani on the development of novel treatments for cancer, parasitic diseases, and brain disorders. In 2016-2017 she again moved to Purdue University as a Temporary Researcher. Dr Brindisi currently holds a position as Assistant Professor at the Department of Excellence of Pharmacy at University of Naples Federico II. A. Prasanth Saraswati obtained his Bachelor in Pharmacy from M.S. Ramaiah College of Pharmacy, Bengaluru, in 2009 and later on obtained his Masters in Medicinal Chemistry from the National Institute of Pharmaceutical Education and Research, Hyderabad, under the supervision of Ahmed Kamal. He is presently pursuing his PhD in Pharmaceutical Sciences at the University of Siena, Italy, as a Marie Curie Early Stage Researcher, mentored by Giuseppe Campiani and Stefania Butini. His research activities encompass the development of novel anticancer agents, including compounds acting through epigenetic mechanism of action, with a special focus on novel therapeutic options for oral squamous cell carcinoma. Simone Brogi is an Assistant Professor at the Department of Pharmacy at University of Pisa. He graduated in 2005 in Biological Sciences from the University of Siena, where he also received his PhD in Pharmaceutical Sciences in 2010. From 2011 to 2019 he has directed the Molecular Modeling Unit at the Department of Biotechnology, Chemistry and Pharmacy (University of



Siena) in the research group of Prof. Giuseppe Campiani. His research activity, described in over 65 papers, is focused on computational approaches for the discovery and optimization of smallmolecules with therapeutic potential for treating cancer and pain, neurodegenerative, cardiovascular, and parasitic diseases. Actually, he is the head of in silico pharmacology and toxicology unit at University of Pisa in the research group of Prof. Vincenzo Calderone. Sandra Gemma is an Associate Professor of Medicinal Chemistry at the University of Siena. She graduated in Chemical and Pharmaceutical Sciences at the University of Siena in 1998 where she also received her PhD degree (2003). She was a post-doctoral fellow at the Department of Chemistry at the University of Illinois at Chicago in the research group of Prof. Arun K. Ghosh. She also visited the Department of Chemistry at Purdue University as a research assistant in the Ghosh group. Her research activity is currently focused on the structure- and ligand-based design and synthesis of therapeutic agents, comprising anti-infective and antiparasitic compounds, anticancer agents and compounds active at the central nervous system. She has authored more than 100 papers in these fields. Stefania Butini is an Associate Professor of Medicinal Chemistry at the University of Siena. She graduated from the University of Siena in 1997 and obtained PhD in pharmaceutical sciences in 2000. From 1999 to 2000, she interned at the University of Groningen, Netherlands, working on the development of novel agents for the treatment of Parkinson’s disease under the supervision of H.W. Wikström. In 2004, she was appointed as senior researcher at the University of Siena. Her research activity (reported in more than 110 manuscripts) includes target selection, rational design of innovative drugs, the development of new synthetic methodologies, and structure-activity relationship studies, with a main focus on CNS diseases, and antiparasitic and antitumor agents.


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Giuseppe Campiani is a full professor of Medicinal Chemistry at Siena University. After his PhD in Pharmaceutical Sciences, Giuseppe Campiani carried out postdoctoral research at Mayo Clinic Jacksonville in the group directed by Prof. Alan Kozikowski and at Columbia University in the City of New York working in the Koji Nakanishi's research group. He was also appointed as visiting professor at Trinity College Dublin. Giuseppe Campiani is presently leading a drug discovery research group at the Department of Excellence of Biotechnology, Chemistry and Pharmacy of Siena University. His broad interest in medicinal chemistry and drug discovery encompasses the development of modulators of epigenetic targets and the discovery of biologically active compounds to combat rare diseases, cancer, neuropsychiatric and neurodegenerative disorders, and infectious diseases.



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Graphical Abstract

Old but Gold: Tracking the New Guise of Histone Deacetylase 6 (HDAC6) Enzyme as a Biomarker and Therapeutic Target in Rare Diseases. Margherita Brindisi, A. Prasanth Saraswati, Simone Brogi, Sandra Gemma, Stefania Butini, Giuseppe Campiani*



Figure 1 111x75mm (600 x 600 DPI)


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Figure 2 193x44mm (600 x 600 DPI)



Figure 3 1276x591mm (96 x 96 DPI)


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Figure4 170x107mm (600 x 600 DPI)



Figure 5 142x115mm (600 x 600 DPI)


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Figure 6 1264x1458mm (96 x 96 DPI)



Figure 7 102x134mm (600 x 600 DPI)


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Graphical Abstract 1363x1348mm (96 x 96 DPI)


HDAC6

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