University of Groningen Deacetylase inhibitors & Histone inheritance Zwinderman, Martijn R. H.

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University of Groningen

Deacetylase inhibitors & Histone inheritance

Zwinderman, Martijn R. H.

DOI:

10.33612/diss.167867692

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Publication date: 2021

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Zwinderman, M. R. H. (2021). Deacetylase inhibitors & Histone inheritance. University of Groningen. https://doi.org/10.33612/diss.167867692

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Chapter 2

Based on:

Martijn R. H. Zwinderman, Fangyuan Cao, Frank J. Dekker.

Acetylation and methylation in asthma, COPD, and lung cancer.

Chemical Epigenetics (2019).

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Acetylation and methylation in asthma, COPD, and

lung cancer

nflammatory pulmonary diseases are among the most common health problems worldwide. Currently, approximately 339 million people suffer from asthma, which results in 250 thousand preventable deaths annually [1, 2]. Similar numbers have been reported for Chronic Obstructive Pulmonary Disease (COPD). About 384 million people were estimated to have COPD and 3 million people died because of it in 2010 [3–6]. COPD is a slow and inevitably progressing disease that, most notably in the later stages, is very debilitating [7]. Inflammatory pulmonary diseases, such as asthma and COPD, are generally chronic and can severely reduce the quality of life. However, until today there is only a limited number of effective therapeutics available for these diseases. In this perspective, novel molecular mechanisms that can be targeted by novel therapeutics need to be identified in order to allow for the development of more sophisticated treatment strategies [1–4, 8]. Inflammation in asthma and COPD is mediated by multiple regulatory mechanisms. On the molecular level, cellular signaling in inflammation is critically regulated by post-translational modifications (PTMs). As an important example, acetylation of histones facilitates transcriptional activation either by neutralizing the ionic interaction between DNA and the histones or by forming a binding site for chromatin remodeling proteins and transcription factors [9, 10]. Disruption of this process results in abnormal gene expression that contributes to the pathogenesis of asthma and COPD. Restoration of protein acetylation thus offers an interesting avenue in the search for new treatments for asthma and COPD. Based on these mechanisms we aim to define the potential utility of histone deacetylase inhibitors (HDACi) as therapeutics in inflammatory pulmonary diseases. For a broader perspective, the role of protein and DNA methylation in these diseases is also described.

I

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Pathogenesis of asthma

Cellular mechanisms of asthma

Asthma is a multifactorial disease with a clear inflammatory component [11]. The pathophysiological mechanisms of inflammation in asthma can broadly be divided into T-helper 2-high (type 2) and T-helper 2-low (non-type 2) subtypes [12]. Hallmarks of T-helper 2 driven inflammation are the presence of the cytokines IL-4, IL-5 and IL-13 and the involvement of mast cells, eosinophils and IgE-producing B cells [13]. These cells influence each other with the mentioned cytokines to mount an inflammatory response. For example, IgE-dependent activation of mast cells triggers them to release their granules containing histamine and tryptase and to generate pro-inflammatory cytokines such as prostaglandin D2, cysteinyl leukotrienes (LTC4 and LTD4) and adenosine [14, 15]. Mast cells are also key sources of the allergy associated cytokines IL-4 and IL-5 [16]. IL-4 aggravates CD4+ T cells to differentiate into T-helper type 2 cells and further enhances IgE-mediated immune responses and inflammatory cell recruitment [17, 18] and IL-5 induces generation of novel eosinophils in the bone marrow and their subsequent chemotaxis towards inflamed lung tissue leading to airway eosinophilia [19]. Eosinophils are the key effector cells that cause most of the tissue damage through release of proteases (Figure 1). Distinctively, where type 2 inflammation can be suppressed by inhalation of corticosteroids, non-type 2 inflammation is largely unresponsive to corticosteroid therapy [20].

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Figure 1. Key cellular mechanisms in asthma and COPD. In asthma, allergen

exposure results in airway remodeling through a persistent type-2 inflammatory response. This response increases eosinophils and damages lung tissue by released proteases from granules (orange dots). Eosinophilic asthma can be suppressed with corticosteroid therapy. In COPD, cigarette smoke results in chronic bronchitis and emphysema through a persistent non-type 2 inflammatory response. This response increases neutrophils and damages lung tissue by released proteases from granules (orange dots) and DNA in neutrophil extracellular traps (NETs). Corticosteroid treatment is largely ineffective in suppressing neutrophilic inflammation [21, 22].

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The molecular and cellular mechanisms that underpin non-type 2 inflammation in asthma are less clear, but may principally involve neutrophils and high levels of IL-17 instead of eosinophils and IL-4, IL-5 and IL-13 [20, 23]. Airway neutrophilia in non-type 2 asthma is the result of an extended lifespan or failed clearance of apoptotic neutrophils and has mostly been observed in severe cases of asthma [24]. As a plausible pathological mechanism, neutrophils form neutrophil extracellular traps (NETs) that may damage nearby lung tissue and impede the resolution of inflammation, Figure 1 [21, 22]. NET formation starts with disassembly of the nuclear envelope, followed by DNA decondensation and rupture of the plasma membrane to expel web-like structures of DNA decorated with histones, proteases and peroxidases [25]. Such NET formation, or NETosis, kills the neutrophil. Additionally, neutrophils are known to degranulate and eject DNA to create NETs extracellularly and leave an anucleated cell body, called a cytoplast, behind [26]. Cytoplasts activate lung dendritic cells to promote differentiation of CD4+ T cells into T-helper 17 cells, which express IL-17 [26]. IL-17 stimulates epithelial cells to produce IL-8 [27] and thus leads to an increase in the production and recruitment of neutrophils, thereby creating a vicious cycle of neutrophil induced lung damage.

Regardless of the type of inflammation, asthma is further pathologically characterized by abnormal structural changes in the airway epithelium and submucosa that lead to airway obstruction, airway hyperresponsiveness and airway mucosal inflammation [28, 29]. The most apparent change in the epithelium is goblet cell hyperplasia [30]. Changes in the submucosa include smooth muscle cell hyperplasia and subepithelial fibrosis [28]. Whether airway remodeling is driven or just modified by inflammation is unclear, but it is clear that inflammation increases the susceptibility to exacerbation of the disease. So, attenuating the persistent expression of inflammatory molecules required for the recruitment and activation of neutrophils, eosinophils and T lymphocytes offers a way to control asthma.

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Role of HDACs in asthma

The acetylation status of proteins in asthma is reported to be maintained by an intricate balance of HATs and HDACs. There are four main classes of HDACs. The class III HDACs use nicotinamide adenine dinucleotide (NAD+) as cofactor. These HDACs are named sirtuins and can reside in the nucleus, mitochondria or cytoplasm depending on the isoform [31]. Class I, II and IV HDACs depend on a zinc ion in their active site for catalytic activity [32, 33]. Class I HDACs, HDAC1, 2, 3 and 8 are generally localized in the nucleus. Class IIA HDACs include HDAC4, 5, 7, 9 and class IIB harbors HDAC6 and 10 and have mostly cytoplasmic functions [34, 35]. Class IV comprises the elusive HDAC11 which is only found in immune cells [36]. The family of proteins that has HAT activity is even more diverse.

HAT activity and the acetylation state of histones is increased in biopsies from children and adults suffering from asthma [37, 38]. Acetylation of histone 3 lysine 27 (H3K27ac) and mono-methylation of H3K4 are essential in maturation of progenitor CD4+ cells into T-helper 2 cells and thus for the pathophysiology of asthma [39]. Additionally, a study found a small decrease in the expression of HDAC1 and HDAC2 in bronchial biopsies of asthma patients compared with healthy individuals [38]. Alveolar macrophages from asthmatic patients are also found to harbor a decreased deacetylase activity related to a decreased expression of HDAC1. The same decrease was not found in circulating blood monocytes thus confirming the notion that aberrant HDAC expression is localized at the site of inflammation [40]. The decreased expression of HDAC1 is also proposed to be a biomarker for severe asthma [41]. The extent of HDAC1 reduction is speculative, since a slight increase in HDAC1 in patients suffering from severe asthma compared with regular patients has also been observed [42]. The expression levels of other HDACs is so far not adequately characterized. In summary, the expression of HDAC1 and HDAC2 in asthma is either slightly increased or decreased and HAT activity is slightly increased.

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Besides differential HDAC expression, single nucleotide polymorphisms in both HDAC1 and HDAC2 have been observed in patients with asthma [43]. A polymorphism in HDAC1 was shown to have a significant relationship with asthma severity and its presence was associated with lung function improvements in response to inhaled corticosteroid treatment in childhood asthmatics [43]. However, the functional role of the polymorphism was not elucidated.

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Pathogenesis of COPD

Cellular mechanisms of COPD

Increased oxidative stress as a result of the inhalation of noxious particles, particularly those derived from cigarette smoke, is a key driving mechanism in the pathogenesis of COPD and leads to the destruction of lung parenchyma [44]. This process is mediated by cytotoxic CD8+ T cells, macrophages and neutrophils. These cells release various proteases, such as elastase and matrix metalloproteinase 9 and 12, that break down connective tissue in the lung parenchyma to result in emphysema [45]. The proteases, together with epidermal growth factors, also induce goblet cell hyperplasia and enhance mucus production and secretion to result in chronic bronchitis [46]. The release of proteases by neutrophils in COPD is, similar to non-type 2 inflammation in asthma, in part the result of NET formation, see Figure 1, mediated by IL-8 [21, 22]. Besides elucidation of the cellular mechanisms, research in COPD has focused at unravelling the intra-cellular pathways that are activated in the inflammatory cells in COPD. In particular, the mechanism behind corticosteroid insensitivity and strategies to revert it have gained attention. These strategies converge at restoring HDAC2 activity, as will be discussed in the next paragraph.

Role of HDACs in COPD

Patients suffering from severe COPD are found to express less than 5% of the HDAC2 than nonsmokers do [47]. This decreased expression of HDAC2, which deacetylates histone 4 (H4) at the IL-8 promotor, correlates with disease progression and is proposed as biomarker for disease severity [48, 49]. Additionally, HDAC5 and 8 were expressed to a lesser extent, SIRT1 activity is decreased, HDAC1, 3, 4, 6 and 7 expression levels were unchanged and the expression and activity of HDAC9, 10, 11 and the other sirtuins is unknown [49, 50]. Altogether, these studies indicate that HDAC expression and activity is altered in COPD.

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Whether the observed decrease in HDAC2 expression in COPD is truly part of the etiology of the disease is difficult to assess. A study that exposed rats to cigarette smoke found a decrease in the expression of HDAC2 after 3 days but not after 8 weeks of cigarette exposure [51]. The initial decrease after 3 days can be the result of a cascade of reactions originating from oxidative stress. Oxidative stress is mediated by a combination of superoxide anions and nitric oxide, derived from cigarettes smoke, to provide peroxynitre [52, 53]. The formed peroxynitre is anticipated to nitrate tyrosine residues on HDAC2 [53]. These nitrated residues then trigger ubiquitination and subsequent proteasomal degradation of HDAC2 [54]. Furthermore, reactive oxygen species activate phosphoinositide-3-kinases, the class I PI3K-δ isoform in particular. The downstream kinase AKT, or PI3K-δ itself, phosphorylates the serine residues on HDAC2, also leading to ubiquitination and proteasomal degradation [55]. This is an explanation for the initial decrease in HDAC2 after 3 days. In the weeks after the initial decrease in HDAC2, synthesis of HDAC2 is upregulated to counteract the proteasomal degradation and to return HDAC2 levels to those found pre-smoke exposure. This would mean that the observed long-term reduction in HDAC2 expression in COPD patients has a different origin.

The group led by Barnes identified HDAC2 as an important regulator of the glucocorticoid receptor (GR) pathway. The GR receptor moves to the nucleus upon activation by a glucocorticoid. There the GR is deacetylated by HDAC2, which allows the GR to form a protein-protein complex that represses the NF-κB pathway [56–58]. This attenuates inflammation in asthma patients taking corticosteroid medication [59]. Conversely, a decrease in the expression of HDAC2, through activation of PI3K-δ during oxidative stress, abolishes the effect of glucocorticoids in patients with COPD [60]. The GR is then not deacetylated and thus cannot repress NF-κB. As further proof, restoration of HDAC2 activity by inhibition of PI3K-δ by either nortryptiline or a low-dose of theophylline helps to alleviate glucocorticoid irresponsiveness in COPD [61–63].

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Interestingly, glucocorticoid irresponsiveness might also result from the upregulation of the beta isoform of the GR. Upon treatment with glucocorticoids, transcription of the GRβ isoform is upregulated, especially in the case of concomitant exposure to IL-17, which is increased in COPD [64– 67]. GRβ directly interferes with the promotor activity of HDAC2 to result in a decreased expression of HDAC2 [68]. This way, the observed reduced level of HDAC2 expression is mainly the result of glucocorticoid treatment, in combination with an increased level of IL17, and not a direct effect of inhalation of noxious particles. This would constitute an acquired form of resistance towards glucocorticoids. The earlier mentioned HDAC expression data in COPD patients taking glucocorticoids was not corrected for the potentially confounding variable of glucocorticoid treatment.

Altogether, the group led by Barnes identified HDAC2 as an important player in activating the anti-inflammatory response upon activation of the GR pathway with glucocorticoids. Additionally, inhalation of noxious particles is linked to an acute decrease in the levels of HDAC2 and these lower levels of HDAC2 might be maintained by continuous smoke exposure and glucocorticoid treatment.

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The role of NF-κB acetylation in asthma

and COPD

The NF-κB signaling pathway plays a central but complex role in asthma and COPD and in inflammation in general. The pathway converges on NF-κB and related transcription factors, which bind to the promotors of pro-inflammatory genes and increase their expression. Importantly, the activity of the NF-κB protein complex is influenced by acetylation. NF-NB contains seven lysine residues that can all undergo the process of acetylation and deacetylation in a site-specific manner. The CBP/p300 acetyltransferases play a major role in acetylating the seven lysine residues on NF-κB [69]. Although HDAC1 and HDAC2 are known to interact with NF-κB, deacetylation of the lysine residues is mostly under the control of HDAC3 [70]. To be more precise, HDAC3 deacetylates lysine 122, 123, 314 and 315, which in their acetylated form negatively regulate NF-κB activity [71]. This is further supported by the finding that HDAC3-deficient macrophages induce only half of the genes linked to LPS-induced inflammatory gene expression [72]. Acetylation of lysines 122 and 123 inhibits binding towards DNA [70]. On the other hand, acetylation of lysines 218 and 221 on subunit p65 (RelA) prevents interaction with the inhibitory protein IκBα, allowing translocation towards the nucleus [73]. Additionally, lysine 310 acetylation is needed for full transcriptional activity of p65, possibly by being a binding site for bromodomain containing proteins that direct transcription [73, 74]. Intriguingly, acetylation of lysines 314 and 315 direct NF-κB towards specific promoter regions [75]. As an example of how acetylation controls the binding site of NF-NB in asthma, the bromodomain protein BRD4 has a binding site for the acetylated NF-κB subunit p65 as well as a binding site for acetylated histones H3k9 and H3k27. That way, BRD4 directs the transcription factor to specific locations along the chromatin to increase the transcription of genes related to proliferation and inflammation [76]. This means that the

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transcriptional activity of NF-κB depends on acetylation. More importantly, the affinity towards different promoters and interaction with transcriptional proteins changes upon acetylation of NF-κB.

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HDACi in asthma and COPD

Currently over 100 HDACi are in clinical trials for cancer therapy [77] and various reports suggest that HDACi could also be effectively used to modulate inflammatory diseases, in part because the underlying disease mechanics in cancer overlap with inflammation [78, 79]. Moreover, the anti-inflammatory effects of HDACi are seen at concentrations 10-100 fold lower than their cell killing properties observed in cancer [80]. Hence, using HDACi for the treatment of inflammatory diseases, like asthma and COPD, is a topic of current research.

Given that HDAC3 is a positive regulator of NF-κB mediated inflammation, inhibitors of HDAC3 have been proposed as novel therapeutics to combat inflammation in COPD and asthma [5]. In support of this, selective inhibition of HDAC3 with the inhibitor RGFP966 in LPS/IFN-γ stimulated macrophages attenuated the NF-kB transcriptional activity and demonstrated anti-inflammatory effects [81]. However, the acetylation status of NF-κB p65, histone H3 and histone H4 was unaffected and the most promising effects were seen at a relatively high concentration of 10 μM, at which RGFP966 also inhibits HDAC1 and HDAC2. On the other hand, siRNA-mediated downregulation of HDAC3 reduced the expression of the pro-inflammatory genes IL-1β, IL-6 and IL-12b up to 60%. This may point to an important structural role for HDAC3 in inflammation instead of its catalytic role in deacetylation. Whether selective pharmacological inhibition of HDAC3 in in vivo models of asthma or COPD will be beneficial remains to be seen. Knocking-out HDAC3 might be a more promising approach.

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Nonetheless, inhibition of HDAC1, 2 and 3 by entinostat (Figure 2) in LPS/IFN-γ induced macrophages in a COPD mouse model led to increased acetylation of NF-κB, increased translocation towards the anti-inflammatory IL-10 promoter and subsequent increased expression of IL-10 [82]. Inhibition of HDAC1, 2 and 3 furthermore reduced inflammation through decreased pro-inflammatory cytokine expression of IL-8, IL-6 and IL-1β. In conclusion, entinostat clearly reduced cigarette smoke-induced airway inflammation in mice and therefore shows potential for the treatment of COPD.

NH₂ H N O N H O O N N O O N H OH N N H N O OH H N O OH N O

Figure 2. Histone deacetylase inhibitors used in in vivo models of asthma or chronic obstructive pulmonary disease.

Besides HDAC1, 2 and 3, HDAC6 and HDAC8 are important in the regulation of cellular processes in inflammation. For example, HDAC8 is known to deacetylate cortactin. This promotes actin filament polymerization and subsequent smooth muscle contraction, which plays an important role in airway inflammation and remodeling [83]. Selective inhibition of HDAC8 with the inhibitor PCI-34051 (Figure 2) has been shown to attenuate airway

Entinostat HDAC1, 2 and 3 Trichostatin A

HDACs

Tubastatin A

HDAC6 PCI-34051HDAC8

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hyperresponsiveness and inflammation and counteract airway remodeling to a certain extent [84]. Another HDAC that is important in airway remodeling is HDAC6, which primary function is the deacetylation of α-tubulin. Tubulin is a major component of the cytoskeleton and is thereby involved in cell motility [85, 86]. Acetylation of α-tubulin leads to stabilized microtubules and thereby decreases cellular motility. The importance of HDAC6 is exemplified by knock-out mouse models that display impeded macrophage migration and motility [87]. Fibroblasts also have reduced motility upon inhibition of HDAC6 [87, 88]. Inhibiting HDAC6 could in this way attenuate airway remodeling in inflammation. Indeed, upon treatment of asthmatic mice with the HDAC6 selective inhibitor tubastatin A (Figure 2), airway hyperresponsiveness and inflammation were mitigated along with a decrease in airway remodeling markers [84]. Additionally, mice that were exposed to cigarette smoke and injected with tubastatin A were protected from cigarette smoke-induced mucociliary clearance disruption [89]. To summarize, HDAC6 and HDAC8 could be targeted with selective HDACi to combat airway remodeling and inflammation in both asthma and COPD.

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T a ble 1 . E ff e ct s of his tone d eac e ty lase inhibit o rs in in vi vo mo de ls o f a sth ma a n d CO PD . T h e pl u s s ign ( + ) ind ic a te s in h ib iti on an d t h e m in u s s ig n (-) in di ca te s n o in h ibi tion of t h e r e sp ect ive h is ton e dea cet y la se is of or m s a t r eleva n t co nce n tr at io ns. In vi vo m o d e l H is to n e d e ac et y la se A st h m a Inhibit o r 1 2 3 6 8 Effe ct Chro ni c asth ma tic m o use m o del [ 84] T u b a st a ti n A - - - + - Reduc e d in fl a m m a ti on P C I-34051 - - - - + Reduced hyperresp on si ven e ss a n d infl am ma tio n Mur in e i n n a te aller g ic lun g in fl a m m a ti on [91] T ric h o st a tin A + + + + + D e cr ea sed a m oun t of in fl a m m a to ry cells a n d i n fl a m m a to ry p ro tein s Ch ro n ic o b str u cti v e pu lm onary d isease C iga re tt e sm oke ex p o sed m ic e [82] En ti n o st a t + + + - - Reduc e d ex p ressi on of IL -8 a n d dec rea sed i n fl ux of n e ut ro ph ils C iga re tt e sm oke ex p o sed m ic e [89] T u b a st a ti n A - - - + - Pro te ct io n fro m ci gare tt e -s m o k e in duc e d m u co ci li ar y c lea ra n ce di srup ti on

33

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More general inhibition of multiple HDACs in in vivo models of airway inflammation also point towards a beneficial anti-inflammatory effect. The non-selective HDACi trichostatin A (TSA, Figure 2), originally reported as a fungistatic antibiotic and one of the first compounds found to inhibit HDACs, reduces inflammation in human precision cut lung slices and in in vivo mouse models [90, 91]. TSA treatment reduced inflammation by reducing the expression of IL-17 and T-helper 17 cell numbers, while increasing Treg cell activation. Furthermore, the expression of TGF-β in the bronchoalveolar lavage fluid was increased following HDAC inhibition in a mouse model of asthma [84]. These changes were associated with increased acetylation at the TGF-β promoter.

The anti-inflammatory effects of TSA have also been postulated to be partly the result of enhanced apoptosis in neutrophils and eosinophils through activation of the c-jun-N-terminal kinase pathway involving caspases 3 and 6 [92]. TSA was shown to have an additive effect on apoptosis in eosinophils following glucocorticoid treatment and TSA antagonized glucocorticoid-induced neutrophil survival [92]. Similar effects have been observed for other non-selective HDACi, which dose-dependently switch neutrophil death from NETosis to apoptosis [93]. This could have important implications for the timely execution of neutrophil apoptosis which has been reported to be dysregulated in severe asthma [24]. Yet, non-selective HDACi also induce apoptosis in macrophages by decreasing the expression of the anti-apoptotic Bcl-2–like protein Bfl-1 in macrophages [94]. Macrophages play an important role in the removal of apoptotic cells [95]. Increased apoptosis of eosinophils and neutrophils without their timely clearance by macrophage will result in disintegration of the apoptotic cells, causing damage to lung tissue and propagation of the inflammatory response [96]. Yet, in addition to macrophages, airway epithelial cells are also capable of phagocytosing apoptotic eosinophils [97] and these epithelial cells, like many other cell-types, do not undergo apoptosis following HDACi treatment [98].

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In conclusion, both isoform selective inhibition as well as more general inhibition of HDACs seem to offer treatment options in asthma and COPD (Table 1). Current investigations aim at defining the importance of specific HDAC isoforms in terms of their structural role or their catalytic role. This provides potential novel starting points for drug discovery. Inhibition of the catalytic activity of HDAC6 and HDAC8 could potentially alleviate airway remodeling and decrease inflammatory cell motility. In contrast, complete knock-out of HDAC3, as described in the beginning of this paragraph, might be required to attenuate airway inflammation.

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Protein and DNA methylation in asthma

and COPD

The role of other PTMs in lung diseases is also being deciphered. Of these other PTMs, most is known about dynamic methylation, governed by methyltransferases and demethylases. Importantly, while reversible acetylation is only known to occur on proteins, both proteins and DNA are found to be methylated. Similar to posttranslational modifications, DNA methylation is a post-replicative modification. However, different enzyme families are at play that control either protein or DNA methylation and demethylation. DNA methyltransferases (DNMTs) methylate the C-5 carbon of cytosines that are next to a guanine, and the presence of 5-methylcytosine (m5C) in a promotor generally leads to a transcriptionally inactive gene [99]. Interestingly, the DNMT inhibitors (DNMTi) azacitidine (Figure 3) and decitabine are substrates for the DNA replication machinery and incorporated into DNA as cytosine substitutes. Once incorporated, they are recognized by DNMTs as if they were natural cytosine, but upon methylation the enzymes are covalently trapped. This leads to the degradation of DNMTs and so ultimately to DNA hypomethylation [100]. Of note, extensive incorporation of azacitidine and decitabine results in DNA damage and apoptosis [101]. Demethylation of DNA on the other hand is a more complicated process. The DNA demethylation pathway begins with oxidation of the methyl group of m5C by the ten-eleven translocation (TET) family of enzymes to result in 5-hydroxymethylcytosine (hm5C), which is then further oxidized to 5-formyl and 5-carboxylcytosine by the same TET enzymes [102–104]. It is currently unclear if the oxidized intermediates serve as epigenetic marks in their own right or if they are just short-lived intermediates in the DNA demethylation pathway. Some evidence indicates that hm5C has some signaling properties, also because it is more prevalent than the formyl and carboxyl derivatives

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N N H N N O O N O HO OH OH N N N NH2 O N H O N H OH OH HO3S SO3H

Figure 3. Selected examples of inhibitors of DNA methyltransferases, lysine demethylases and protein arginine methyltransferases.

[105]. In any case, the final product in the oxidiation cascade, 5-carboxylcytosine, could in theory be decarboxylated to reform unmodified cytosine. However, a decarboxylase that is capable of decarboxylating 5-carboxylcytosine is yet to be identified. Known mechanisms to date show that the oxidized versions of 5mC are diluted in a replication-dependent manner to regenerate unmodified cytosine or in the case of formyl and 5-carboxylcytosine are recognized and excised by enzymes involved in base excision repair mechanisms [106, 107]. The first step in this process is to flip the oxidized base out of the DNA double helix followed by cleavage of the N-glycosidic bond, which is catalyzed by thymine DNA glycosylase [108]. This leaves a gap in the DNA while leaving the sugar phosphate backbone intact. Other enzymes then refill the gap with an unmodified cytosine.

Protein demethylation is a somewhat simpler process that nonetheless also starts with oxidation [109]. However, in this case oxidation results in an unstable intermediate, a hemiaminal, which degrades to release formaldehyde and the thus demethylated lysine or arginine residue. Similar to HDACs, lysine demethylases (KDMs) come in various isoforms, and KDMs are grouped based on their dependency on either Fe(II) and 2-oxoglutarate or flavin [110–112]. In contrast, so far just one enzyme, the Jumonji domain containing protein 6, is speculated to have arginine demethylation activity in vivo [113]. The functionally opposing group of methyltransferases for both

Azacitidine DNMTs

GSK-J4

KDM6 PRMTsAMI-1

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lysines and arginines is even larger and more diverse. Additionally, while acetyltransferases catalyze the addition of just one acetyl group to a lysine residue, methyltransferases mono-, di-, or tri-methylate them and mono- or di-methylate arginines [114]. The extent of methylation and the specific residues involved dictate the functional outcome.

Cross-talk between acetylation and methylation

An additional layer of complexity is added by the fact that PTMs can be linked mechanistically by processes that are collectively termed “cross-talk” [115]. As an example, methylation of histone H3 on lysine 4 (H3K4) recruits acetyltransferases to acetylate several other lysine residues of histone H3 [116]. Furthermore, the degree to which H3K4 is methylated directly influences the extent of H3 acetylation [117]. As a result, trimethylation of H3K4 is generally considered to be a marker of transcriptional activation. Additionally, methylation of H3K4 is known to inhibit DNA methylation, thereby further ensuring transcriptional activation [118, 119]. Conversely, DNA methylation decreases H3K4 methylation and H3 acetylation through a group of proteins that specifically recognize and bind to methylated DNA and in turn recruit a protein complex that contains HDACs and histone demethylases [120, 121]. Cross-talk thus not only occurs between histone modifications but also between histone modifications and other epigenetic processes like DNA methylation. Not surprisingly, cross-talk between PTMs of many different proteins exists, simply because lysines are found to be both acetylated and methylated, and these modifications affect protein functionality in different ways. It is often not clear how these different modifications compete with each other.

DNA methylation in asthma and COPD

There does not seem to be a clear association between DNA methylation levels and the presence of COPD. While there are studies that suggest that DNA methylation may be a biomarker of COPD [122], a genome-wide analysis of DNA methylation using blood samples of 903 never and 658 current smokers from the general population failed to show any significant association [123]. A

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systematic review of six articles that assessed the association of COPD with DNA methylation also failed to see consistency in the data [124]. Perhaps due to this lack of a clear link between DNA methylation and COPD, no inhibitors of DNA methyltransferase have been tested so far in in vivo models of COPD. Interestingly, the promotor of HDAC6 is known to be hypomethylated in COPD [89], which may partly explain the elevated HDAC6 expression seen in COPD and further point to treatment with HDAC6 selective inhibitors. In contrast, a large meta-analysis of DNA methylation in childhood asthma, using data from more than 5,000 children, identified reduced DNA methylation levels in 14 distinct sites to be associated with asthma across childhood from ages 4 to 16 years [125]. Interestingly, whole blood DNA methylation profiles were strongly driven by lower methylation within eosinophils, further highlighting the important role of eosinophils in asthma. Additionally, the association was not found at birth, suggesting that environmental factors, like allergen exposure, could be the main cause of the observed change in the DNA methylation status. This possibility is further supported by results from mouse models of asthma, which showed reduced overall m5C and increased hm5C that correlated with a respective decrease in DNMTs and increase in TET enzymes upon allergen exposure [126, 127]. This indicates that DNMT activity limits asthma severity and thus that increasing DNA methylation may be beneficial in asthma. In stark contrast, treatment with azacitidine, a non-selective DNMTi, has been reported to reduce inflammation and airway hyperreactivity in mice, possibly by increasing the numbers of regulatory T cells [128]. Future research therefore needs to determine whether treatment of asthma with DNMTi or with still to be developed DNA demethylase inhibitors will be most beneficial.

Protein methylation in asthma and COPD

There is limited data on the role of arginine and lysine methylation in asthma and COPD. With respect to arginine methylation, one study reports the expression levels of protein arginine methyltransferase (PRMT) isoforms 1 to 6 in an animal model of asthma [129]. Except for PRMT4, the expression of all

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other PRMTs was found to be increased. In asthmatic rats, PRMT1 expression was especially high in epithelial cells [130]. The epithelial cells also expressed and secreted more eosinophil-attracting chemokines and fibroblast-activating cytokines. Consequently, the fibroblast proliferated more and expressed more growth factors, most likely through IL-1β-induced NF-κB activation [131, 132]. Interestingly, PRMT1 expression was also increased in the fibroblasts. Furthermore, treatment of the asthmatic rats with AMI-1 (Figure 3), a non-selective inhibitor of PRMTs, ameliorated the observed pulmonary inflammation, mucus secretion and collagen generation. This indicates that inhibition of PRMTs might be a valuable therapeutic strategy in asthma. Another reason for PRMT inhibition in asthma is that methylated arginine in itself, resulting from metabolic turnover of methylated proteins, is known to inhibit nitric oxide synthase [133, 134]. This results in a decrease in nitric oxide and an increase in reactive oxygen species, through which methylated arginine potentiates lung inflammation and airway hyperresponsiveness. In contrast to asthma, the expression of PRMT6 is downregulated in lung tissue of COPD patients, as well as in mice with emphysema [135]. Furthermore, restoration of PRMT6 expression in mice exposed to cigarette smoke extract resulted in a decrease in inflammation, apoptosis and oxidative stress. PRMT6 expression was restored by treatment with a lentivirus that was encoded with the PMRT6 gene. The protective effect of PRMT6 expression indicates that COPD treatment should focus on increasing protein methylation. However, the role of the other PRMT isoforms and protein demethylases in COPD is as of yet unknown. Consequently, the outcome of treatment of COPD with either PRMT or demethylase inhibitors is unpredictable. As with HDAC inhibition, the extent of inhibition of specific isoforms of PRMTs or demethylases will dictate the outcome. The field is thereby in need of the discovery and testing of isoform-selective inhibitors of PRMTs and demethylases in models of COPD.

In comparison to arginine methylation, less is known about the functional role of lysine methylation in asthma and COPD. This is in spite of the fact that

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potent and selective lysine demethylase inhibitors have been developed [136, 137]. One in vitro study found that reduced lysine histone methylation enhances the expression of vascular endothelial growth factor in airway smooth muscle cells from patients with asthma, thereby playing a role in airway remodeling [138]. An in vivo study found that restoration of lysine methylation by treatment with the potent KDM6 inhibitor GSK-J4 (Figure 3) ameliorated the classical hallmarks of asthma, such as airway hyperresponsiveness, airway inflammation and remodeling [139]. Treatment with GSK-J4 did however not decrease the expression of the vascular endothelial growth factor. The main explanation for the observed positive effects of GSK-J4 is that the inhibitor decreased the proliferation and migration of airway smooth muscle cells and prevented the upregulation of contractile proteins in these cells. In conclusion, in asthma, an increase in lysine methylation and a decrease in arginine methylation have so far shown to be potentially beneficial treatment strategies. For COPD nothing is known about the role of lysine methylation, but the role of arginine methylation in COPD is starting to be explored. The first studies are showing a beneficial effect of the restoration of arginine methylation in COPD.

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HDACi and DNMTi in lung cancer

Of the different types of inhibitors described in this chapter, primarily HDACi and DNMTi are in clinical trials for lung cancer. Alterations in DNA methylation and protein acetylation are both considered to be major contributors to the development and progression of lung cancer. Lung cancer is generally classified as either small-cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), with the latter category split into a range of other histological subtypes. NSCLC accounts for approximately 85% of all lung cancer cases and SCLC takes up the remaining 15% [140]. Most SCLC tumors initially respond to chemotherapy, but, unfortunately, essentially all patients experience relapse within 1 year of receiving first-line treatment. Similar to COPD, smoking is the major risk factor for all forms of lung cancer, particularly for SCLC.

DNMTi and HDACi have both demonstrated anticancer effects in in vitro NSCLC studies. However, single-agent clinical trials with these inhibitors in lung cancer patients mostly failed to show a beneficial effect. The current view is that the combination of DNMTi or HDACi with established therapies might increase efficacy by priming cancer cells to standard chemotherapy, possibly by reactivation of tumor suppressor genes [141]. Consequently, DNMTi and HDACi are in a host of phase I and II clinical trials in combination therapies, for instance with immune checkpoint inhibitors like the monoclonal anti-PD1 antibody nivolumab. A detailed description of these trials can be found elsewhere [142].

Interestingly, a direct link between HDACs and DNMTs exists, since it has been found that HDACs deacetylate DNMTs [142]. In support of this, knockdown of HDACs and treatment with a HDACi induced DNMT acetylation. Surprisingly, this led to a decrease in DNMT levels. Overall, treatment with HDACi may thus concomitantly decrease DNMT levels. Since increased expression of DNMTs and HDACs crucially occurs in the

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oncogenic transformation of epithelia it is therefore speculated that HDAC inhibition may prevent lung cancer. Additionally, combinations of DNMTi and HDACi are under investigation because they may synergize in the re-expression of silenced genes. A phase I/II trial of low-dose azacitidine combined with entinostat in 45 extensively pretreated patients with recurrent metastatic NSCLC showed that the combination was well tolerated and objective responses were observed [143]. This included one complete responder that was free of disease 26 months since enrolment. The combination also compared favorably with erlotinib, the existing treatment option. However, the objective responses to this therapy occurred in only 4% of patients and the median survival in the entire cohort was nevertheless just 6.4 months. Two other phase II trials using the combination of azacitidine and entinostat in lung cancer are still ongoing [144]. The results of these trials will hopefully clarify the potentially synergistic effect between HDACi and DNMTi.

Finally, an interesting ongoing phase I clinical trial investigates the efficacy of inhaled azacitidine in patients with advanced NSCLC [144]. Generally, azacitidine is administered by subcutaneous injections, leading to systemic exposure. However, localized delivery of the drug to the lungs by means of inhalation was proven highly effective in animal models, because it was associated with longer survival, less toxicity, and less lung cancer burden compared to subcutaneous injection [145]. The results of that study sparked the initiation of the phase I trial. The outcome of this trial will be interesting as it only investigates the effect of inhaled azacitidine, not in combination with other therapies, which as a single-agent therapy has so far been unsuccessful. A caveat for the use of inhaled therapies in lung cancer is that between 30 and 40% of patients will have systemic metastases at the time of clinical diagnosis [146]. Logically, a therapy localized to the lungs will then need to be combined with a more systemic one.

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Conclusion

Progress has been made in the chemotherapy of lung cancer by employing drugs that influence the reversible posttranslational and post-replicative modifications of proteins and DNA respectively. However, optimum treatment regimens with such drugs still need to be defined, and it is clear that mostly a combination with another drug is required. In that sense the triple therapy of a HDACi, a DNMTi and an immune checkpoint inhibitor is an interesting approach that is currently being investigated. Additionally, other influencers of PTMs, like inhibitors of KDMs, are in clinical trials for lung cancer [144]. It is to be expected that in the future an even broader range of compounds that influence the state of PTMs will be studied in combination therapies for lung cancer.

The results of the studies in lung cancer are highly relevant to other lung diseases like asthma and COPD, because one way to view the airway remodeling observed in asthma and COPD is to compare it to benign tumor growth. For instance, in both cases, cell proliferation is out of control and tissue boundaries are weakened. Therefore, as in lung cancer therapy, using drugs that regain control of tissue proliferation and architecture might be promising in asthma and COPD. Yet, as mentioned in this chapter, the study of HDACi and other influencers of PTMs in these diseases is still in the proof-of-principle stage. The initial in vivo preclinical studies are nonetheless promising, especially in the case of HDACi. In mouse models of asthma and COPD, application of several HDAC inhibitors has shown to result in a broad range of beneficial effects. Local administration of HDACi at relatively low concentrations may therefore hold great promise for the treatment of these diseases.

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