Histone deacetylases and the immunological network ... - Nature

Oncogene (2010) 29, 157–173

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REVIEW

Histone deacetylases and the immunological network: implications in cancer and inflammation A Villagra1,2,3,4, EM Sotomayor2,3,4 and E Seto1,4 1 Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL, USA; 2Department of Immunology, H Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL, USA and 3Department of Malignant Hematology, H Lee Moffitt Cancer Center and Research Institute at the University of South Florida, Tampa, FL, USA

The initiation, magnitude and duration of an immune response against antigens are a tightly regulated process involving a dynamic, orchestrated balance of pro- and anti-inflammatory pathways in immune cells. Such a delicate balance is critical for allowing efficient immune response against foreign antigens while preventing autoimmune attack against self-antigens. In recent years, much effort has been devoted to understanding immune evasion by cancer cells. Also, significant advances have been made in mechanistically understanding the role of pro- and anti-inflammatory cytokines in the regulation of immune responses against antigens, including those expressed by tumors. However, we still know very little about the regulation of inflammatory/anti-inflammatory genes in their natural setting, the chromatin substrate. Several mechanisms have been identified to influence chromatin flexibility and allow dynamic changes in gene expression. Among those, chromatin modifications induced by acetylation and deacetylation of histone tails have gained wide attention. In this study, we discuss the role of histone deacetylases in the transcriptional regulation of genes involved in the inflammatory response and how these enzymes coordinate the dynamic expression of these genes during an immune response. This emerging knowledge is opening new avenues to better understand epigenetic regulation of inflammatory responses and providing new molecular targets for either amplifying or ameliorating immune responses. Oncogene (2010) 29, 157–173; doi:10.1038/onc.2009.334; published online 26 October 2009 Keywords: HDAC; inflammation; cytokine regulation

Correspondence: Drs A Villagra or EM Sotomayor or E Seto, Department of Molecular Oncology, H Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA. E-mails: alejandro.villagra@moffitt.org or eduardo.sotomayor@moffitt.org or ed.seto@moffitt.org 4 These authors contributed equally to this study. Received 31 July 2009; revised 5 September 2009; accepted 13 September 2009; published online 26 October 2009

Introduction Multiple mechanisms are involved in gene regulation. Among these, the availability of regulatory factors and chromatin structure have important roles. Regulatory factors become available through cellular localization, gene expression and post-translational regulation. Chromatin structure is determined by covalent and non-covalent modifications of the DNA itself (for example, DNA methylation and destabilization of the double helix) or by changes in proteins associated with the DNA (for example, acetylation, methylation and phosphorylation). Histones constitute one family of DNA-associated proteins, and are often targeted for numerous covalent and non-covalent modifications that ultimately affect the state of chromatin structure. For example, acetylation of positively charged amino acids is one such covalent modification. Although acetylation is mediated by histone acetyltransferases, removal of the acetyl group is catalysed by histone deacetylases (HDACs). Modification by these enzymes is not restricted to histones; non-histone proteins, including transcription factors, can also be regulated. Thus, given their dual functionality to influence chromatin structure and transcription factors, histone acetyltransferases and HDACs are at the center of gene regulation. Histone deacetylases are enzymes that are often recruited by corepressors or multi-protein transcriptional complexes to gene promoters, in which they regulate transcription through chromatin modification without directly binding DNA. Eighteen HDACs have been identified and divided into four classes (Yang and Seto, 2003): Class I includes HDAC1, 2, 3 and 8; Class II includes HDAC4, 5, 6, 7, 9 and 10; Class III consists of members of the sirtuin family of HDACs; and Class IV is represented by HDAC11, the newest family member discovered (Gao et al., 2002). Although histones are a primary substrate for HDACs, several studies have shown that HDACs also deacetylate nonhistone proteins such as p53, E2F1, RelA, YY1, TFIIE, BCL6 and TFIIF (Glozak et al., 2005). Furthermore, although this review will focus on the role of HDACs in gene expression, increasing evidence suggests that some HDACs seem to have a wide range of protein

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substrates in addition to histones and factors relevant to transcription. Histone deacetylases are the target of several structurally diverse compounds known as HDAC inhibitors (HDIs) (Marks et al., 2000). Of note, these compounds were used as inhibitors long before a clear understanding of the role of specific HDACs in normal and/or transformed cells began to emerge. These compounds can induce cytodifferentiation, cell cycle arrest and apoptosis of transformed cells (Marks et al., 2000; Bolden et al., 2006). Moreover, some HDIs have showed significant antitumor activity in cancer patients, including one compound, SAHA, which has been approved by the Food and Drug Administration for the treatment of patients with cutaneous T-cell lymphomas (Mann et al., 2007; Marks and Breslow, 2007). In spite of these important therapeutic advances, the underlying target(s) and/or mechanism(s) mediating the antitumor activity exhibited by HDIs in human malignancies remain to be fully elucidated. The HDAC involvement and the therapeutic use of HDI are not restricted to cancer, as several studies have also shown that these enzymes have a role in autoimmune diseases (Pankaj et al., 2008), inflammatory regulation (Blanchard and Chipoy, 2005), central nervous system disorders (Kazantsev and Thompson, 2008) and development (Haberland et al., 2009). Interestingly, most of the intracellular pathways involved in these processes share common intermediaries that are regulated by HDACs, suggesting a central role for these enzymes as regulators of seemingly unrelated physio-pathological conditions. Supporting this concept, HDIs have emerged as potential therapeutic tools for the treatment of autoimmune diseases (Mishra et al., 2001; Pankaj et al., 2008), cystic fibrosis (Rubenstein and Zeitlin, 1998) muscular dystrophy (Minetti et al., 2006) and regulation of immune tolerance (Tao and Hancock, 2007). In contrast to the rapidly increasing knowledge of the role of HDACs in cancer and the use of HDIs in treating this and other pathological conditions, still little is known about the role of specific HDACs in immune cells and the functional consequences of their inhibition by HDIs. Although some studies have highlighted the ability of HDIs to augment inflammatory and antitumor responses (Blanchard and Chipoy, 2005), others have shown the opposite, that is, HDIs displaying antiinflammatory properties and amelioration of graftversus-host disease and autoimmune disorders (Reddy et al., 2004; Sandra et al., 2005; Glauben et al., 2006). Thus, elucidation of the underlying target(s) and mechanism(s) involved in these divergent effects of HDIs remain a challenge given the pan-inhibitory effect of the compounds currently in use. To complicate things even further, there is accumulating evidence that the role of specific HDACs in immune cells extends beyond histones and now encompasses more complex regulatory functions that are dependent on the enzyme’s tissue expression profile, cellular compartment distribution, stage of cellular differentiation and/or pathophysiological conditions (Glozak et al., 2005; Minucci and Pelicci, Oncogene

2006). For instance, HDAC6 is localized mainly in the cytoplasm and is uniquely endowed with tubulin deacetylase activity (Hubbert et al., 2002). This HDAC is a key regulator of the cytoskeleton, cell migration and cell–cell interactions (Valenzuela-Fernańdez et al., 2008). Moreover, in immune cells, HDAC6 has a role in the formation of the antigen-presenting cell (APC)/ T-cell immune synapse (Serrador et al., 2004). Similarly, HDAC11 is a transcriptional repressor of interleukin (IL)-10 gene expression in APCs (Villagra et al., 2009) whose expression has been found to be tissue-restricted to the brain, kidney and testis, as well as abnormally overexpressed in certain types of leukemias and lymphomas (Gao et al., 2002). In this review, we first discuss current knowledge of the role of specific HDACs in the transcriptional regulation of pro- and anti-inflammatory cytokines in immune cells. Then, we highlight the emerging regulatory role of HDACs in immunological networks participating in inflammatory responses. Finally, we discuss the changes in HDACs that occur in immune cell-derived tumors, such as leukemias and lymphomas, and how the use of HDIs may potentially tip the balance toward effective antitumor responses due to their effects on tumor cells and the host’s immune cells. As there are several excellent recent reviews that discuss the topic of Class III HDACs (sirtuins) in immune response and inflammation (Salminen et al., 2008; Finkel et al., 2009; Natoli, 2009), this review will focus more on the Class I and II enzymes. HDACs and cytokine regulation Cytokines are low molecular weight proteins or glycoproteins secreted by cells of the immune and other systems in response to a variety of extracellular stimuli. Typically produced de novo, these molecules exert their functions during a very narrow period of time (Cohen and Cohen, 1996). Cytokines can be classified according to their structural homology, receptor binding capabilities, immune response and cellular sources. Much evidence points to these molecules playing a central role in the regulation of inflammatory responses. Cytokines displaying pro- or anti-inflammatory effects have been identified and the kinetics of their production, magnitude and concentration at the site of the immune reaction are considered as main determinants in shaping the initiation, intensity and duration of an immune response (Cavaillon, 2001). For instance, IL-12 and 10, cytokines with divergent inflammatory properties, are critical in regulating these dynamic changes and maintaining a delicate balance to prevent autoimmune attack while allowing an efficient immune response against foreign antigens (Moore et al., 2001; Trinchieri, 2003; Li and Ra, 2008). Cytokine gene expression can be up- or downregulated in response to specific stimuli. Such regulation involves several intracellular signaling pathways and occurs very rapidly to affect the cytokine’s transcriptional rate. Of note, a common feature among these


159 Table 1

Participation of HDACs in the transcriptional regulation of cytokines

HDAC

Protein target

Gene target

Comment

HDAC1 HDAC7, 9

Sp1 FOXP3

IL-1 IL-2

HDAC1

ZEB1

IL-2

HDAC1 HDAC1

— GR

IL-12 IL-5

HDAC5

—

IL-8

HDAC2

GR

GM-CSF

HDAC1, 6, 8

—

IFN-b

HDAC1, 2

Sin3A

Ifng

HDAC11

—

IL-10

HDAC1, 2, 3

—

IL-4

HDAC3

Pro-IL-16

Skp2

HDAC1

STAT5, C/EBPb

Id-1

HDAC3 HDAC1, 2, 3

STAT3 STAT3

STAT3 target genes STAT3 target genes

HDAC3

STAT1

STAT1 target genes

HDAC3, 4, 5

GATA-1

GATA-1 target genes

HDAC3, 5

GATA-3

GATA-3 target genes

HDAC3

GATA-2

GATA-2 target genes

HDAC3

NF-kb

NF-kb target genes

Sp1 recruits HDAC1 to IL-1 promoter HDAC7 and 9 interact with FOXP3 and TIP60 to repress IL-2 expression ZEB1 recruits CtBP and HDAC1 to the IL-2 promoter HDAC1 is recruited to IL-12 promoter GR binds to IL-5 promoter and recruits HDAC1, interfering with GATA3 positive regulation HDAC5 is recruited to IL-8 promoter in cells infected with Legionella pneumophila Glucocorticoid receptor recruits HDAC2 to GM-CSF promoter to antagonize the effect of IL-1b HDAC1 and 8 repress IFN-b expression, and HDAC6 acts as a coactivator to enhance activity Sin3A Recruits HDACs to the Ifng locus for repress its transcription in Th0 cells HDAC11 is recruited to IL-10 promoter to repress transcription HDACs are recruited to IL-4 promoter to repress transcription HDAC3 binds to pro-IL-16 to repress Skp2 expression and subsequently promote cell cycle arrest STAT5 recruits HDAC1 to Id-1 promoter for C/EBPb deacetylation, allowing transcriptional activation HDAC3 enhances STAT3 phosphorylation HDACs deacetylate STAT3 to inactivate its transcriptional functions HDAC3 deacetylates STAT1 allowing its phosphorylation and nuclear translocation Transcriptional regulation mediated by GATA-1 is diminished in the presence of HDACs Phosphorylated GATA-3 recruits HDAC3 and HDAC5 to specific DNA regions HDAC3 suppresses the transcriptional potential of GATA-2 RelA is deacetylated by HDAC3 inducing its nuclear export

Reference Enya et al. (2008) Li et al. (2007) Wang et al. (2009) Lu et al. (2005) Jee et al. (2005) Schmeck et al. (2008) Ito et al. (2000) Nusinzon and Horvath (2006) Chang et al. (2008) Villagra et al. (2009) Valapour et al. (2002) Zhang et al. (2008) Xu et al. (2003) Togi et al. (2009) Yuan et al. (2005) Kra¨mer et al. (2009) Watamoto et al. (2003) Chen et al. (2006) Ozawa et al. (2001) Chen et al. (2001)

Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; HDAC, histone deacetylase; IFN, interferon; IL, interleukin; NF-kB, nuclear factor-kB.

different pathways is that they are regulated by molecules promoting the appropriate transcriptional microenvironment at the level of the chromatin substrate. Among these molecules, HDACs influence gene transcription by modifying the cytokine’s promoter regions. The ability of HDACs to also modify nonhistone proteins, including transcription factors such as STAT3, further highlights the important role of these enzymes in cytokine gene regulation (Yuan et al., 2005). Indeed, cytokine production by immune cells has been shown to be regulated by changes in the acetylation status of their promoters (Yao et al., 2005; Zhang et al., 2006). The involvement of HDACs in cytokine regulation is not restricted to any specific class or subtype of cytokines. In fact, HDACs appear to participate in the transcriptional regulation of both pro- and anti-inflam-

matory cytokines. Table 1 summarizes our current knowledge regarding HDAC regulation of cytokines, as well as the target proteins enabling HDAC recruitment to specific DNA sequences. HDACs and regulation of proinflammatory cytokines Inflammation occurs in response to pathogens, irritants, damaged cells and other stimuli. This response has been also implicated in the initiation and progression of cancer (de Visser and Coussens, 2005; Khan and Tomasi, 2008). For instance, some malignancies may be prevented by the use of anti-inflammatory drugs (Anderson et al., 2006; Rigas, 2007). Inhibition of certain proinflammatory cytokines has also been shown to decrease the aggressiveness of some tumor types Oncogene


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(Blanchard and Chipoy, 2005). A better understanding of the signal-transduction pathways and transcription regulators leading to inflammation will likely provide important hints toward which pathways and biological processes (for example, apoptosis and/or cell cycle control) become deregulated in cancer. For example, IL-8 is a proinflammatory cytokine produced rapidly in response to infection, enabling recruitment of granulocytes and macrophages to the injured site (Strieter, 2002). At the molecular level, the IL-8 promoter is rapidly acetylated by p300, a process that is accompanied by a transient decrease in HDAC1 and HDAC5 within its promoter region. At later time points, however, these HDACs are recruited back to the IL-8 promoter most likely to terminate transcriptional activity (Schmeck et al., 2008). Interestingly, IL-8 has recently been shown to be overexpressed in several malignancies including breast cancer (Freund et al., 2003), and its aberrant expression favors angiogenesis as well as the growth and invasiveness of malignant cells (Lin et al., 2004). Whether this dysregulation of IL-8 in cancer cells is the result of changes in the expression/ function of p300, HDAC1 or HDAC5 remains to be determined. Two other proinflammatory cytokines controlled by HDACs are IL-12 and IL-1. IL-12 is regulated at the transcriptional level by histone acetylation. This modification is mediated by p300 to promote gene activation. Conversely, HDAC1 has been shown to repress IL-12 gene expression (Lu et al., 2005). IL-1 has been postulated to increase tumor invasiveness and metastasis in various experimental models (Dinarello, 1996). IL-1a gene expression is repressed by a complex consisting of HDAC1 and Sp1 protein in melanoma cells (Enya et al., 2008). Such modulation of IL-1a expression has been proposed to influence the aggressiveness of this malignancy (Onozaki et al., 1985). Histone deacetylases have also been reported to modulate IL-2, a key cytokine involved in the development, differentiation and homeostasis of T cells. Expression of this cytokine is inhibited strongly by HDAC1 in cooperation with the transcriptional repressor zinc-finger E-box-binding protein (ZEB)-1 and the corepressor C-terminal-binding protein (CtBP)-2 (Wang et al., 2009a). IL-2 expression has been shown to be deregulated in certain viral and autoimmune diseases as well as in some leukemias (Gesbert et al., 1998). A better understanding of the molecular mechanisms and the consequences of HDAC1-mediated repression of IL-2 gene expression will not only provide important clues to the observed IL-2 deregulation but may also unveil novel targets for overcoming T-cell anergy, a state of unresponsiveness characterized by lack of IL-2 production in response to cognate antigen (Kametani et al., 2008). FOXP3 is an additional repressor of IL-2 gene expression that directly associates with the acetyltransferase protein TIP60 (Tat-interacting protein, 60 kDa), HDAC7 and HDAC9. HDAC7 and HDAC9 associate with FOXP3 under different conditions, suggesting that they may have different roles in regulating IL-2 gene expression (Li et al., 2007). Oncogene

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced by immune cells in response to several stimuli. Production of GM-CSF is abrogated following treatment with low concentrations of glucocorticoids (Barnes, 2005). HDAC2 has been reported to have a role in this steroid-induced inhibition of GMCSF given: (a) the ability of this HDAC to interact with the glucocorticoid receptor (GR) and (b) the rapid recruitment of HDAC2 to the GM-CSF promoter (as observed by chromatin immunoprecipitation) when cells are treated with dexamethasone (Ito et al., 2000). Furthermore, deacetylation of GR by HDAC2 enables interaction between the receptor and nuclear factor (NF)-kB, promoting its repressor activity upon GMCSF expression (Ito et al., 2006). Finally, IL-5 is a cytokine that has a central role in allergic reactions through eosinophil activation. Glucocorticoids are effective drugs for the treatment of several allergic conditions. Recruitment of HDAC1 by GR to the IL-5 promoter has been shown to repress transcription of this gene. Two potential mechanisms have been proposed to explain this HDAC1-mediated effect: (a) recruitment of GR with HDAC1 to the DNA may alter the ability of the transcriptional activator GATA3 to bind its DNA target sequence and activate IL-5 gene expression or (b) the HDAC1–GR complex may promote a non-permissive chromatin structure for GATA3 binding (Jee et al., 2005). HDACs and regulation of anti-inflammatory cytokines In addition to regulating the expression of proinflammatory cytokines, recent evidence has demonstrated that HDACs have important roles in controlling the transcription of anti-inflammatory cytokines. IL-10 is one of the best characterized cytokines and is involved in tolerance induction and inhibition of inflammatory responses. Recently, we demonstrated that HDAC11 downregulates IL-10 transcription in murine and human APCs by interacting at the chromatin level with the distal region of the promoter (positions 807–1653, relative to the transcription initiation site). Such an effect not only determines the inflammatory status of these cells but also influences priming versus tolerance of antigen-specific CD4 þ T cells (Villagra et al., 2009). Mechanistically, we found that overexpression of HDAC11 in APCs strongly inhibited IL-10 transcriptional activity. Chromatin immunoprecipitation analysis of these cells revealed a lack of histones H3 and H4 acetylation at the IL-10 proximal promoter (positions 1–807) that was associated with diminished recruitment of STAT3 and Sp1. These data most likely reflected the decreased accessibility of these transcription factors to less acetylated, and therefore more compact, chromatin. Importantly, we confirmed the requirement for HDAC11 enzymatic activity in this process, as overexpression of an HDAC mutant with a truncated deacetylase domain failed to inhibit IL-10 gene expression. Instead, an increase in IL-10 mRNA was observed, perhaps as a result of the HDAC11 mutant acting as a


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dominant negative variant and competing with endogenous HDAC11. Similar enhancement in IL-10 gene expression was observed when HDAC11-specific short hairpin RNAs were expressed in APCs. In these cells, we observed increased H3 and H4 acetylation that was accompanied by enhanced recruitment of Sp1 and STAT3 to the proximal region of the IL-10 promoter. These changes, together with the absence of negative regulation in the distal promoter mediated by the transcriptional repressor PU.1 and/or HDAC11, provide a plausible explanation for the enhanced IL-10 gene expression observed in HDAC11-deficient APCs. The significance of these findings lies at several levels. First, a physiological role for HDAC11 has been provided. Second, HDAC11, by inducing dynamic changes at the chromatin level, regulates the expression of IL-10 and possibly other genes involved in the inflammatory response. This effect may explain, at least partially, the plasticity of the APC to determine T-cell activation versus T-cell tolerance. Third, HDAC11 represents a novel molecular target to potentially influence immune activation versus immune tolerance, a critical decision with significant implications in cancer immunotherapy, transplantation and autoimmunity. Class I HDACs have been reported to inhibit IL-4 expression in T cells (Valapour et al., 2002). However, the mechanisms involved in the recruitment of these HDACs to the IL-4 promoter are unknown. Nevertheless, their repressive action on IL-4 expression has raised considerable interest in the potential manipulation of Class I HDACs for the treatment of asthma (Choi et al., 2005). HDAC1 and 2 have also been implicated in the transcriptional control of genes regulated by tumor necrosis factor-a. For instance, these HDACs are recruited to the chitinase YKL-40 (human cartilage glycoprotein 39) promoter to repress its transcription. Tumor necrosis factor-a in a process that could be mediated through NF-kB is mainly responsible for HDAC recruitment to this promoter (Bhat et al., 2008). Of note, YKL-40 has been shown to induce migration of endothelial cells as well as proliferation and protection against inflammatory damage in a variety of cells (Ling and Recklies, 2004). Another anti-inflammatory cytokine, interferon-b (IFN-b), is induced in response to some viral infections or double-stranded RNA. This induction is dependent upon the orchestrated action of several transcriptional regulators, including different HDACs (Chang et al., 2004). Interestingly, although HDAC1 and 8 are negative regulators of IFN-b expression, HDAC6 acts as an essential coactivator that induces its transcription (Nusinzon and Horvath, 2006). This example demonstrates that different HDACs may regulate the same gene and have opposing roles.

HDACs and cytokines involved in T-cell differentiation Naive CD4 T cells differentiate into two types of effector cells, Th1 or Th2. Both subsets differ in their

cytokine profile and function during an immune response. Some representative cytokines produced by Th1 and Th2 cells are IFNg and IL-4, respectively. In addition, the most effective cytokines driving this differentiation are IL-12 for Th1 and IL-4 for Th2 (Bowen et al., 2008; Aune et al., 2009). Importantly, all cytokines involved in the differentiation of Th1 and Th2 cells are regulated by HDACs, highlighting their central role in this process. One of the best examples of HDAC participation in T-cell differentiation is the epigenetic regulation of the IFNg locus. Most changes in DNA methylation and acetylation of the IFNg locus occur within a conserved non-coding sequence >50 kb both upstream and downstream of the Ifng gene (Zhou et al., 2004), illustrating the importance of the three-dimensional chromatin structure of this locus in its regulation (Aune et al., 2009). Association of HDAC1 and 2 with Sin3A can lead to the formation of the transcriptional corepressor complex Sin3 (Yang and Seto, 2008). The Sin3/HDAC1/2 complex is constitutively associated with the IFNg locus in Th0 cells, thereby repressing the expression of this cytokine in naive T cells. Following differentiation of Th0 cells into Th1 cells, this repression is relieved by displacement of Sin3/HDAC complexes. This process is tightly regulated by Th1-related transcriptional regulators such as T-Bet, which promote Sin3/HDAC complex dissociation (Chang et al., 2008), histone acetyltransferase activity, and, ultimately, chromatin acetylation. The IFNg and T-Bet genes are highly acetylated in Th1, but not Th2 cells, and this hyperacetylation is directly associated with their expression. Interestingly, when Th2 cells are treated with HDI, the acetylation status of both genes is recovered even though their gene expression is not restored, suggesting a more complex mechanism involved in Th1/Th2 differentiation (Morinobu et al., 2004). A similar mechanism regulates the IL-4 gene locus in T-cell differentiation. When naive T cells are polarized to the Th2 phenotype, they acquire specific H3K9/14 acetylation (Ansel et al., 2006) that correlates with enhanced expression of IL-4. In contrast, only a slight increase in acetylation of the IL-4 locus is observed in Th1 cells (Avni et al., 2002). Interleukin-5 and 13 are two additional cytokines expressed in Th2 cells. Both are regulated at the transcriptional level by GATA3 and GR (Zhang et al., 1998; Jee et al., 2005), which have been shown to interact with HDACs (Chen et al., 2006; Ito et al., 2006). A current model proposes that GR associates with the IL-5 proximal promoter, where it binds NF-AT and AP-1. This binding impairs GATA3 recruitment to the promoter and activates IL-5 expression. In addition, GR recruits HDACs to the local promoter region, favoring a more condensed chromatin conformation (Jee et al., 2005). Interleukin-2 also has an important role in the development and differentiation of T cells (Gesbert et al., 1998). HDAC1 is a strong repressor of IL-2 expression in these cells, and its specific activity on the IL-2 promoter is dependent upon the presence Oncogene


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of transcription factors such as ZEB1 and CtBP2 (Wang et al., 2009a). Of special interest is the inhibition of IL-2 in response to bacterial superantigens, a process that is tightly associated with the ability of HDAC1 to bind the IL-2 promoter (Kametani et al., 2008). Finally, HDACs also participate in regulating T-cell survival. For instance, HDAC7 is a known repressor of the orphan nuclear receptor Nur77, inhibiting T-cell receptor (TCR)-mediated apoptosis (Dequiedt et al., 2003). Nur77 is expressed in T cells immediately after TCR activation to promote negative selection (Woronicz et al., 1994). Overexpression of Nur77 is associated with apoptosis, whereas its absence inhibits TCR-mediated cell death (Calnan et al., 1995). The functionality of HDAC7 seems to be regulated by reversible phosphorylation, shuttling this deacetylase to the cytoplasm after TCR-dependent phosphorylation (Parra et al., 2007). Cross-regulation of HDACs and immunological networks There is special interest in the participation of HDACs in the regulation of STAT and NF-kB signaling and their relationship to autoimmunity and cancer. HDACs not only directly activate/repress these pathways but are also modulated by these signaling pathways themselves. In this section, we will discuss the role of HDACs in the regulation of these important processes, as well as the influence of STAT and NF-kB signaling intermediaries in regulating HDAC nuclear localization and degradation. HDACs in the STAT and NF-kB pathways Histone deacetylases associate with multiple proteins to form many different constitutive stable complexes, including the Sin3, Mi-2/NuRD, CoREST and N-CoR/SMRT complexes (Yang and Seto, 2008). However, HDACs also associate with proteins in a tissue- or stimulus-specific manner. For example, in unstimulated cells, the p50 subunit of NF-kB binds HDAC1 to repress the transcription of NF-kB target genes. After activation, the p50/p65 heterodimer translocates to the nucleus and displaces p50/HDAC1. This mechanism explains the negative role of p50 on transcription (Zhong et al., 2002). A similar mechanism has been proposed for HDAC3, in which it associates with TAB, N-CoR and p50 (Baek et al., 2002). HDAC3 also deacetylates lysine 221 of RelA (p65) in the nucleus, promoting NF-kB binding to IkBa and its nuclear export (Chen et al., 2001). This process terminates NF-kB activity and recycles protein components to the cytoplasm for new rounds of activation. In addition, acetylation of RelA at lysine 310 is required for its transcriptional activity, through an IkBa-independent mechanism, indicating that acetylation of this protein regulates different functions (Chen et al., 2002). Phosphorylation of STATs is required for their activation, nuclear translocation and transcriptional Oncogene

regulation of target genes (Ihle, 2001). STAT1 is phosphorylated in response to cytokine and growth factor stimulation. This event could be counteracted by dephosphorylation or CBP acetylation, which promotes STAT1 nuclear export (Kra¨mer et al., 2006). Once acetylated, STAT1 can be deacetylated by HDAC3, as well as HDAC1 and 2, thus permitting its phosphorylation and reactivation (Klampfer et al., 2004; Kra¨mer et al., 2009). Although HDACs promote STAT1 activation and nuclear import, their effects on STAT3 is exactly the opposite (Figure 1). STAT3 is acetylated by p300 in response to cytokine treatment (Paulson et al., 1999), and this deacetylation is reversed by Class I HDACs, leading to impairment of STAT3 dimerization and subsequent transcriptional regulatory activity (Yuan et al., 2005). A recent study has shown HDAC3 interaction with phosphatase 2A (Togi et al., 2009), suggesting the existence of a more complicated scaffold of modifying proteins, where phosphorylation and acetylation coordinately regulate each other. STAT5 is also regulated by HDACs. This transcription factor is an activator of the Id-1 gene, a dominant negative inhibitor of basic helix-loop-helix transcription factors such as E2A, HEB and E2-2 (Massari and Murre, 2000), and a key regulator of B- and T-cell functions (Engel and Murre, 2001). By recruiting HDAC1 to the Id-1 promoter to deacetylate C/EBPb, STAT5 can bind DNA and promote Id-1 transcription (Xu et al., 2003). Regulation of HDACs by immunological signaling Histone deacetylases are regulated many different ways. The magnitude, selectivity and timing of their regulation are dictated by cellular context and the specific combination of events affecting the target cell. The activities of almost all HDACs are regulated by protein–protein interactions and the composition of the participating protein complexes. In addition, HDACs are regulated by post-translational modification, subcellular localization, transcriptional regulation, proteolytic processing and cofactor availability. Class I HDACs are expressed in many different tissues, whereas Classes II and IV are expressed in a more tissuespecific manner (de Ruijter et al., 2003). However, regardless of these physiological variations of HDAC levels, they can be regulated at the transcriptional level by different intra- and extracellular signals, including HDI (Hauser et al., 2002; Bradbury et al., 2005), lipopolysaccharide (LPS) (Aung et al., 2006), phytohemagglutinin (Dangond and Gullans, 1998) and cytokines (Bartl et al., 1997). In Figure 2, three possible mechanisms for regulating HDACs by immunological pathways and signaling are illustrated. Subcellular localization and proteolytic processing are key elements in many immune signaling networks, including the NF-kB (Gopal and Van Dyke, 2006) and MAPK pathways (Baek et al., 2002). In addition, expression of several HDACs can be altered by bacterial components. For example, Legionella


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Figure 1 Functional roles for histone deacetylases (HDACs) in the transcriptional regulation of cytokines. (A) Direct recruitment of HDACs mediated by transcription factors binding to specific DNA sequences. (B) Deacetylation of transcriptional mediators for activation and subsequent nuclear translocation. (C) Deacetylation of transcriptional mediators for nuclear export. (D) Deacetylation of transcriptional mediators for inactivation, which is frequently associated with loss of DNA-binding capability. Light blue arrows indicate mobilization of protein/complexes.

Figure 2 Regulation of histone deacetylases (HDACs) by immunological pathways. Three possible mechanisms for HDAC regulation: (A) Proteosomal degradation; (B) intracellular shuttling; and (C) transcriptional regulation. Nuclear factor (NF)-kB signaling can be activated through the classical tumor necrosis factor (TNF)-a pathway. This cytokine binds to its receptor to trigger nuclear import of the NF-kB heterodimer and subsequent activation of survival and proinflammatory genes. NFkB can also be activated by interferon (IFN)g, lipopolysaccarides (LPS) and interleukin (IL)-1b, all of which converge on a common mediator, the IkB kinase complex. The subunit IkB kinase complex (IKKb) from this complex is responsible for the ubiquitination of HDAC1. Red arrows indicate different pathways involved, and light blue arrows show mobilization of protein/complexes. Oncogene


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pneumophila infection can increase the expression of HDAC5 but not other HDACs (Schmeck et al., 2008). LPS stimulation of macrophages affects the expression of almost all HDACs, albeit to different magnitudes and kinetics (Aung et al., 2006). For example, HDAC6, 10 and 11 are gradually downregulated up to 8 h after LPS stimulation with a slight recovery after 24 h. The same expression pattern is observed for HDAC4, 5 and 7; however, these deacetylases are upregulated 24 h after LPS stimulation. In contrast, HDAC1 is gradually upregulated until 8 h, and becomes stabilized by 24 h after stimulation. These changes in HDAC expression in response to LPS stimulation have prompted the use of selective HDI for controlling the immune response, as these enzymes participate in the expression of pro- and anti-inflammatory mediators. Proinflammatory signaling such as those activated by LPS, IL-1b, tumor necrosis factora and IFNg induces HDAC1 ubiquitination and proteosomal degradation. This process involves the same intermediaries as NF-kB signaling (Gopal and Van Dyke, 2006). Once these proinflammatory molecules bind to their respective receptors, their signals converge onto a common pathway that leads to activation of NF-kB-inducing kinase and, subsequently, the IkB kinase complex (IKK). IKK induces the phosphorylation and ubiquitination of IkBa, enabling nuclear import of the NF-kB heterodimer. A subunit of the IKK complex, IKKb, is responsible for the ubiquitination of HDAC1 (Gopal et al., 2006). HDACs also regulate the intracellular localization of NF-kB and MAPK signaling components. For instance, HDAC3 associates with TAB2 and N-CoR proteins in a corepressor complex recruited by the p50 subunit of NF-kB. This complex translocates from nucleus to the cytoplasm upon IL-1 stimulation through MEKK1, which phosphorylates TAB2 to expose its nuclear export signal (Baek et al., 2002). This mechanism is not restricted to HDAC3 as HDAC1 has also been demonstrated to interact with p50 (Zhong et al., 2002). However, physical interaction between TAB2 and N-CoR has not been reported for HDAC1 complexes. Regulation of HDAC availability through intracellular localization control seems to be a general event. For instance, the cellular localization of HDAC7 is regulated by reversible phosphorylation. In inactive T cells, HDAC7 is located in the nucleus and represses several genes involved in T-cell differentiation, including Nur77 (Dequiedt et al., 2003). After TCR activation, the serine/ threonine kinase PKD1 phosphorylates HDAC7, thus promoting its cytoplasmic accumulation. Conversely, the myosin phosphatase complex (PP1b/MYPT1) dephosphorylates HDAC7, promoting its nuclear accumulation and subsequent inhibition of its target genes (Parra et al., 2007). HDAC7 has also been shown to be cleaved by caspase-8 in T cells tagged by extrinsic apoptotic pathways (Scott et al., 2008). Modification of HDAC activity is another regulatory mechanism mediated by cytokines. In this context, A375-R8 melanoma cell lines expressing IL-1a constitutively have been shown to decrease HDAC1 activity Oncogene

(Enya et al., 2008). Because HDAC activity has been hypothesized to be reduced by oxidative stress (Barnes et al., 2004), it is possible that this biological condition may be present in A375-R8 cells, leading to decreased HDAC activity. In addition, HDAC2 activity is reduced by tyrosine nitration in response to oxidative stress (Ito et al., 2004; Osoata et al., 2009), and expression of this HDAC is decreased in smokers (Ito et al., 2001) and patients with chronic obstructive pulmonary disease (Ito et al., 2005). Finally, pan HDI has been reported to influence the expression of some HDACs. Specifically, acute myeloid cells treated with different HDI show increased HDAC11, 9 and SIRT4 expression (Bradbury et al., 2005). Another interesting finding is the synergistic action of HDI with phytohemagglutinin for upregulating HDAC1 and 3 expression in T cells (Dangond and Gullans, 1998). HDACs in tumors arising from immune cells (hematologic malignancies) Epigenetic changes in cancer cells are well-documented phenomena, and there is much interest in understanding the exact detailed epigenetic mechanisms in cancer. For example, loss of histone H4-K16 acetylation and histone H4-K20 trimethylation are distinguishing features in many cancers. Importantly, these events appear early during tumor initiation (Fraga et al., 2005). An additional hallmark of tumor cells is DNA methylation. Global hypomethylation and promoter CpG island hypermethylation are frequent alterations observed in cancer (Feinberg and Vogelstein, 1983; Esteller, 2002). These findings highlight the major role of epigenetic modifiers in cancer. Three major mechanisms have been identified in the initiation and progression of cancer, namely the activation of silenced genes and/or production of fusion proteins, the silencing of tumor suppressor genes and the repression of genes involved in the antitumor response. Currently, there is much interest in understanding the mechanisms governing the regulation of immune genes in cancer (Khan and Tomasi, 2008), such as the involvement of microRNAs (miRNAs) in the altered gene regulation in cancer. Data indicate that 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites (Sevignani et al., 2006), and that miRNAs are involved in the regulation of many immune-related genes, including major histocompatibility complexes (MHC) and costimulatory molecules (Asirvatham et al., 2009). This hypothesis proposed by Khan and Tomasi (2008) suggests that miRNAs are silencing specific genes by binding to nascent RNA transcripts, a process that could be mediated by HDACs as shown at heterochromatic foci (Tomari and Zamore, 2005). Another possibility is alteration of the cytokine balance in tumor cells, and deregulation of the cellular machinery responsible for tagging cancer cells for immune recognition and elimination. For instance, control of MHC expression can be mediated by cytokines that are


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regulated tightly by epigenetic modifiers, including HDACs. Role of HDACs in modulating the immune response against cancer T cells recognize antigenic peptides presented on the surface of host cells by MHC molecules, of which there are two types, Class I (MHC-I) and Class II (MHC-II). Both are expressed differentially. Thus, MHC-II is only found on professional APCs such as dendritic cells, B cells and macrophages. MHC-I is found on all nucleated cells and mainly presents peptides of endogenous proteins to CD8 þ cytotoxic T cells. Peptides presented by MHC are recognized by T cells, which facilitate the elimination of cells carrying non-self peptides derived from endogenous pathogens or mutated proteins, thereby providing an important protection against virus infection and tumor mutations. Much effort has been devoted to understanding immune evasion by cancer cells, and how MHC regulation affects the initiation and progression of cancer. MHC generation and regulation are key steps to consider. In particular, the mechanisms involved in this process and how intracellular pathways influence antigen presentation in cancer cells must be understood. Mutations and aberrant expression of MHC-I and components of the antigen processing and presentation machinery (APM) have been found in different cancers, including hematological malignancies (Seliger, 2008). For instance, b2-microglobulin has been shown to be mutated or deleted in colon carcinoma, melanoma and other tumors (Seliger et al., 2006). Also, the deregulation of several other components of the MHC-I APM, occurring at the transcriptional and post-transcriptional levels, has been observed (Aptsiauri et al., 2007; Cabrera et al., 2007; Seliger, 2008). Regulation of MHC-I expression seems to be dependent on the aggressiveness of tumors and progression of cancer as it has been shown that lower levels of MHC are related to poor prognosis in different malignancies (Connor et al., 1993), and metastatic tumors have lower expression of MHC Class I than primary tumors (Cromme et al., 1994). The loss or downregulation of MHC-I and/or APM components allows tumor cells to evade the immune system. Identifying the mechanisms by which their expression is regulated will help discover candidates for future treatments against cancer. In this context, cytokines have an important role in the regulation of MHC and APM components. For example, IFNg, tumor necrosis factora, GM-CSF and IL-4 are able to upregulate the expression of MHC and APM components (Petersson et al., 1998; Hallermalm et al., 2001; Guo et al., 2002). Moreover, IL-10 can repress some APM components (Zeidler et al., 1997). Thus, HDACs could be participating indirectly in the regulation of these molecules through their capacity to modulate the expression of regulatory cytokines. In addition, epigenetic modifiers can induce the expression of MHC-I and APM components. Several reports show

that MHC Classes I and II are repressed by DNA methylation in numerous malignancies (van den Elsen et al., 2003; Karpf, 2006), and that the treatment of tumor cells with 5-azacytidine upregulates the MHC expression in cancer cells (Serrano et al., 2001; Karpf et al., 2004). Direct evidence of HDAC participation in the expression of MHC-I and II has been reported. In particular, HDAC1 and 2 repress MHC-II expression in human cancer cervical cell lines (Zika et al., 2003), whereas HDAC1, 2 and 8 repress MHC-I in these cells (Maeda et al., 2000; Magner et al., 2000; Li et al., 2006). The use of HDI also increases the expression of MHC-I, -II and different APM components and costimulatory molecules in several types of cancer cells (Khan and Tomasi, 2008). In addition, it has been reported that HDI promotes the expression of MHC Class I-related chains A and B, making cancer cells susceptible to elimination by natural killer cells (Skov et al., 2005; Zhang et al., 2009). Aberrant expression of HDACs and fusion proteins in cancer Alterations in HDAC expression are found in several types of cancer. For example, HDAC1, 2 and 3 are overexpressed in ovarian and endometrial carcinomas (Weichert et al., 2008a), prostate (Weichert et al., 2008b) and renal cancer (Fritzsche et al., 2008). HDAC1 is overexpressed in cancers of the breast (Zhang et al., 2005), colon (Wilson et al., 2006) and pancreas (Wang et al., 2009b), whereas HDAC3 expression is increased in colon cancer (Wilson et al., 2006). HDAC7 is overexpressed in pancreatic cancer (Ouaı¨ ssi et al., 2008), and HDAC8 expression is deregulated in neuroblastoma (Oehme et al., 2009). The list of aberrant HDAC expressions in cancer continues to grow and, in almost all of them, there is a correlation between high expression and poor prognosis. However, there are exceptions. For instance, HDAC6 overexpression occurs in breast cancer and is associated with a better prognosis (Zhang et al., 2004). The silencing of specific HDACs is also associated with reduced aggressiveness, proliferation and cancer survival (Bolden et al., 2006). Also, expression of some Classes II and IV HDACs are decreased in neuroblastoma, one of the most lethal malignant brain tumors (Lucio-Eterovic et al., 2008). Only a few hematological malignancies show changes in HDAC expression. Some subtypes of diffuse large B-cell lymphoma exhibit increased HDAC1 expression (Poulsen et al., 2005). In a recent study, HDAC1, 2 and 6 expression in diffuse large B-cell lymphoma and peripheral T-cell lymphoma was higher than in normal lymphoid tissue (Marquard et al., 2009). In this report, it was suggested that HDAC6 overexpression correlated with a favorable outcome in diffuse large B-cell lymphoma patients, whereas the opposite effect was observed in peripheral T-cell lymphoma patients. Most irregularities affecting HDACs in hematological malignancies are associated with the presence Oncogene

Oncogene

x

x

x

x

x

x

x

x

x

SAHA (Vorinostat) Hydroxamate

PXD101 (Belinostat) Hydroxamate

FK228 (Depsipeptide) Cyclic peptide

MGCD0103 Benzamide

MS-275 (SNDX-275) (Entinostat) Benzamide

x

x

x

LBH589 (Panobinostat) Hydroxamate

2

1

HDACi

x

x

x

4

x

x

o

o

NA o

x

x

x

3

x

x

x

6

o

NA o

o

NA o

x

x

x

5

o

NA o

o

x

x

x

8

x

x

x

x

10

NA o

o

x

x

x

9

Ongoing studies in combination with: Bortezomib (proteosome inh.) 17-AAG (Hsp90 inh.) Decitadine (hypomethylating agent) Etoposide (topoisomerase II inh) Rituximab (CD20 ab)

Ongoing studies in combination with: Bortezomib (proteosome inh.) 17-AAG (Hsp90 inh.) Imatinib (Tyr. Kinase inh.) Lenalidomide

Combinational drugs

Ongoing studies in combination with: Gemcitabine (nucleoside analog) 5-azacitidine (hypomethylating agent)

NA Ongoing studies in combination with: Azacitidine (hypomethylating agent) Sargramostin (GM-CSF)

x

x

x

x

11

Ongoing studies in combination with: Bortezomib (proteosome inh.) 17-AAG (Hsp90 inh.) Azacitidine (hypomethylating agent) NA NA NA NA NA Ongoing studies in combination with: Bortezomib (Proteosome inh.) Decitadine (Hypomethylating agent) Rituximab (CD20 ab)

x

x

x

7

Table 2 Examples of HDAC inhibitors in clinical trials

Feng et al., 2007; Gimsing et al., 2008; Gimsing, 2009; Ma et al., 2009

Furumai et al., 2002; Lech-maranda et al., 2007; Hartlapp et al., 2009; Zhou and Zhou, 2009

Induction of oxidative stress and DNA damage in transformed cells. Enhances proapoptotic pathways.

Activation of intrinsic and/ or extrinsic apoptotic pathways. Induction of Hsp90 hyperacetylation. Inhibition of angiogenesis by downregulation of VEGF expression.

No proposal for possible mechanisms. However, Antiproliferative activity, cell cycle arrest, and increased apoptosis has been observed in transformed cells. Generation of reactive oxygen species in transformed cells. Enhances proapoptotic pathways.

Open: 9 (6 hematological malignancies) Completed: 8 (4 hematological malignancies) Open: 10 (6 hematological malignancies) Completed: 27 (10 hematological malignancies)

Open: 0 Completed: 7 (7 hematological malignancies)

Open: 7 (3 hematological malignancies) Completed: 5 (4 hematological malignancies)

Dokmanovic et al., 2007; Marks and Breslow, 2007; Al-Janadi et al., 2008; Emanuele et al., 2008; Frew et al., 2009

Induce changes in the expression of pro- and antiapoptotic proteins, activating the intrinsic and/or extrinsic apoptotic pathways. Possible mechanisms involving cell proliferation and migration.


Acute myeloid leukemia (AML). Phase II Hodgkin lymphoma. Phase II

Giles et al., 2006; Ellis et al., 2008; Atadja, 2009; Frew et al., 2009

Decrease chaperone function of Hsp90, leading to destabilization and degradation of growth factors and their downstream signalings, that is, Bcr-Abl, Flt-3, c-Raf.


Cutaneous T-cell lymphoma (CTCL). Phase II chronic myelogeneous leukemia (CML). Phase III acute myeloid leukemia (AML). Phase II Hodgkin’s lymphoma. Phase II Multiple myeloma (MM). Phase I Cutaneous T-cell Lymphoma (CTCL). Approved Chronic myelogeneous leukemia (CML). Phase III Acute myeloid leukemia (AML). Phase II Hodgkin’s Lymphoma. Phase II Multiple myeloma (MM). Phase III Diffuse large B-Cell lymphoma (DLBCL). Phase II Mantle cell lymphoma (MCL). Phase II Chronic lymphocytic leukemia (CLL). Phase II T-Cell Lymphoma. Phase II Acute Myeloid Leukemia (AML). Phase II Non-Hodgkin Lymphoma. Phase II Multiple Myeloma (MM). Phase III Cutaneous T-cell lymphoma (CTCL). Phase II Acute Myeloid leukemia (AML). Phase II Non-Hodgkin’s Lymphoma. Phase II Multiple myeloma (MM). Phase II Diffuse large B-Cell lymphoma (DLBCL). Phase II Mantle cell lymphoma (MCL). Phase II Chronic Lymphocytic leukemia (CLL). Phase II Acute Lymphoblastic lymphoma (ALL). Phase I Acute myeloid leukemia (AML). Phase II Non-Hodgkin’s lymphoma. Phase II Hodgkin lymphoma. Phase II

Rosato et al., 2003; Hess-Stumpp et al., 2007; Nishioka et al., 2008

Bonfils et al., 2008; Fournel et al., 2008; Tourneau et al., 2008

References

Possible mechanisms

Clinical trials (NCI, April 2009)

Hematological malignancies


166

Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; HDAC, histone deacetylase; NA, information not available; o, no inhibition; VEGF, vascular endothelial growth factor; x, inhibition.

Kuendgen and Gattermann, 2007; Duenas-Gonzalez et al., 2008 Enhances extrinsic and intrinsic proapoptotic pathways. Open: 16 (8 hematological malignancies) Completed: 14 (5 hematological malignancies) Acute myeloid leukemia (AML). Phase III Non-Hodgkin’s lymphoma. Phase I Chronic lymphocytic leukemia (CLL). Phase II x x Valproic Acid Aliphatic acid

x

x

x

x

o

x

x

o

NA Ongoing studies in combination with: Decitabine (hypomethylating agent) ATRA (all-trans retinoic acid) Etoposide (topoisomerase II inh.)

References Possible mechanisms Clinical trials (NCI, April 2009) 10 1 HDACi

Table 2 Continued

2

3

4

5

6

7

8

9

11

Combinational drugs

Hematological malignancies


167

of chimeric oncoproteins where aberrant fusion proteins recruit corepressor-containing HDACs to inhibit the expression of specific target genes, that is, t(15;17)(q22;q21) PML-RARa (Redner, 2002), t(11;17)(q23;q21) PLZFRARa (Redner, 2002) and AML1-ETO (Durst and Hiebert, 2008). The consequence of these translocations is the creation of a new transcriptional regulatory framework. Thus, the repression of key genes involved in cell proliferation and apoptosis control, such as the tumor suppressor p14ARF (Linggi et al., 2002), blocks differentiation and inhibition of apoptosis (So and Cleary, 2004). Another consequence is the downregulation of genes involved in specific hematopoietic differentiation, such as the colony-stimulating factor-1 (Follows et al., 2003). Non-Hodgkin’s lymphomas also show different translocations involving cell cycle and apoptotic regulators. Some documented cases follow (Pasqualucci et al., 2003): lymphoplasmacytic lymphoma, the translocation t(9;14)(p13;q32), which involves the proto-oncogene Pax-5, a transcription factor regulating B-cell proliferation and differentiation; follicular lymphoma, the translocations t(14;18)(q32;q21), t(2;18)(p11;q21) and t(18;22)(q21;q11), all of which involve Bcl-2, a negative regulator of apoptosis; mantle cell lymphoma, t(11;14)(q13;q32), involving Bcl-1, which encodes for the cell cycle regulator cyclin D1; MALT lymphoma, t(11;18)(q21;q21) and, rarely, t(1;14)(p22;q32), involving Ap12/Mlt and Bcl-10, respectively; Ap12 has antiapoptotic activity, which is also a probable action of Bcl-10; in diffuse large B-cell lymphoma, t(3;other)(q27;other), involving Bcl-6, a transcriptional repressor required for germinal center formation; Burkitt’s lymphoma, t(8;14)(q24;q32), t(2;8)(p11;q24) and t(8;22)(q24;q11), involving c-Myc, a transcription factor regulating cell proliferation and growth; anaplastic large T-cell lymphoma, t(2;5)(p23;q35), involving Npm/Alk, where Alk is a tyrosine kinase. Interestingly, most of the genes involved in these translocations are also regulated by HDACs, thereby increasing the possibility of using HDI as therapeutics for treating these malignancies. HDI in the treatment of hematological cancers Histone deacetylase inhibitors are a heterogeneous group of chemical compounds that inhibit HDAC enzymatic activity. They are classified by their chemical structure into six different groups: short-chain fatty acids (for example, valproic acid), hydroxamates (for example, trichostatin A, SAHA), benzamides (for example, MS-275), cyclic tetrapeptides (for example, depsipeptide), electrophilic ketones (for example, trifluoromethylketone) and miscellaneous compounds (for example, MGCD0103). The inhibitory effect of HDIs is due to their interaction with the active zinc site in the catalytic pocket of HDACs, as determined by crystallographic studies of the HDAC8/hydroxamate complex (Somoza et al., 2004). On the basis of their antiproliferative capabilities, HDIs were classified as anticancer drugs in erythroleukemic cells (Leder et al., Oncogene


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1975). However, the molecular mechanism behind their functional properties was discovered years later when HDAC inhibition was observed following trichostatin A treatment of several cell lines, including mammary gland tumor cells (Yoshida et al., 1990). Histone deacetylase inhibition can result in the induction of apoptosis through extrinsic (for example, death receptor) and intrinsic (for example, mitochondria) pathways, cell cycle arrest, induction of differentiation, antiangiogenic, anti-invasive and immunomodulatory functions (Bolden et al., 2006; Minucci and Pelicci, 2006; Dokmanovic et al., 2007). Interestingly, most of these effects are seen in transformed cells, whereas normal cells appear to be more resistant to HDI (Dokmanovic et al., 2007). The exact mechanisms that allow normal cells to tolerate higher concentrations of HDI than transformed cells are not completely understood. However, results suggest that a protective effect of thioredoxin against damage by reactive oxygen species may be the key (Shao et al., 2004), as cancer cells lack this protein and normal cells with thioredoxin knockdown accumulate reactive oxygen species and display increased cell death (Ungerstedt et al., 2005). An important number of HDIs have progressed to clinical trials for individual use or in combination with other drugs and/or radiation. Table 2 lists some of the

most important HDIs and their use in clinical trials. The wide spectrum of hematological malignancies under investigation with HDI, and the promising results obtained with some of them alone or in combination with other drugs, are notable (Bhalla, 2005; Bishton et al., 2007; Al-Janadi et al., 2008; Rasheed et al., 2008). However, the use of pan-HDIs lacking specificity does not allow for a definitive mechanistic explanation regarding the action of these compounds on malignancies. Nevertheless, some investigators propose that their effect is mediated mainly by repression of Class I HDACs, as demonstrated by the use of HDIs and specific small interfering RNAs against Class I HDACs (Glaser et al., 2003; Inoue et al., 2006). According to this hypothesis, the use of pan-HDIs could be improved by using the selective compounds that do not target or have a lesser effect on Class II HDACs, such as MS-275 (Class I selective) and MGCD0103 (Classes I, IV and HDAC10 selective). Without a doubt, the use of selective HDIs will generate more comprehensive information about the mechanisms of these compounds in treating cancer, providing more accurate effects to be assigned to simple targets and/or pathways. We must remember that the use of HDIs for treating cancer not only affects the proliferation and survival of cancer cells, but also other pathways, including immunological networks.

Figure 3 Differential targeting of histone deacetylases inhibitors (HDI). The final action of HDIs on the expression of particular genes is dependent on multiple factors. For instance, HDIs have different IC50 values for individual HDACs, and in some cases, their action is restricted to only a few of them. Also, HDACs targeted by HDIs are expressed differentially according to the cell type. In most situations, the expression of HDACs is altered under non-physiological conditions, such as cancer and autoimmune diseases. Finally, the effect of an HDI on any gene could be either direct or indirect and mediated by another protein targeted by the compound. Oncogene


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Conclusions The pathways and regulators involved in controlling the immune response have captured the interest of immunologists and researchers in other areas including cancer biology, development and cell cycle control. More recently, HDACs have been shown to have pivotal roles in the regulation of immunological pathways. Interestingly, the activity of HDACs is not exclusive to the transcription level; they also regulate non-histone proteins, giving a new perspective on the mechanistic action of these enzymes. In this context, HDI are a potential tool for controlling the immune response during infection, transplantation, autoimmune diseases and cancer. However, the participation of HDACs in these processes is not completely understood. Moreover, some information accumulated thus far is contradictory and incomplete. For example, HDAC inhibition can enhance the immune response in some cases and repress it in others. This duality could be explained by the differential effect of HDACs on pro- and anti-inflammatory cytokines, as well as the cellular context. Also, considering that HDIs have different preferences for particular HDACs, we could argue that the differing outcomes observed by HDIs are due to their effects on

different HDACs. Figure 3 depicts a potential model for the action of HDIs on specific genes. This model supports combinatorial steps that ultimately lead to the repression or activation of particular genes. The specificity of HDIs, along with the expression profile of HDACs under normal and non-physiological conditions, contributes to the final effects of these compounds. Also, indirect effects of HDIs on secondary or tertiary proteins that could potentially activate or repress target genes should be considered. The combinatorial model for the action of HDIs must take into consideration the cell type, cellular context, physiopathological condition and HDAC preference. Conflict of interest The authors declare no conflict of interest. Acknowledgements The work in our laboratories was supported by grants to ES from the National Institutes of Health (NIH), the American Heart Association and the Kaul Foundation, and to EMS from the NIH.

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