Oncogene (2010) 29, 157–173 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc REVIEW deacetylases and the immunological network: implications in cancer and inflammation

A Villagra1,2,3,4, EM Sotomayor2,3,4 and E Seto1,4

1Department 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 Introduction response against antigens are a tightly regulated process involving a dynamic, orchestrated balance of pro- and Multiple mechanisms are involved in regulation. anti-inflammatory pathways in immune cells. Such a Among these, the availability of regulatory factors and delicate balance is critical for allowing efficient immune chromatin structure have important roles. Regulatory response against foreign antigens while preventing auto- factors become available through cellular localization, immune attack against self-antigens. In recent years, and post-translational regulation. much effort has been devoted to understanding immune Chromatin structure is determined by covalent and evasion by cancer cells. Also, significant advances have non-covalent modifications of the DNA itself (for been made in mechanistically understanding the role of example, DNA methylation and destabilization of pro- and anti-inflammatory cytokines in the regulation of the double helix) or by changes in proteins associated immune responses against antigens, including those with the DNA (for example, acetylation, methylation expressed by tumors. However, we still know very little and phosphorylation). constitute one family about the regulation of inflammatory/anti-inflammatory of DNA-associated proteins, and are often targeted in their natural setting, the chromatin substrate. for numerous covalent and non-covalent modifications Several mechanisms have been identified to influence that ultimately affect the state of chromatin structure. chromatin flexibility and allow dynamic changes in gene For example, acetylation of positively charged amino expression. Among those, chromatin modifications acids is one such covalent modification. Although induced by acetylation and deacetylation of histone tails acetylation is mediated by histone acetyltransferases, have gained wide attention. In this study, we discuss the removal of the acetyl group is catalysed by histone role of histone deacetylases in the transcriptional regula- deacetylases (HDACs). Modification by these tion of genes involved in the inflammatory response and is not restricted to histones; non-histone proteins, how these enzymes coordinate the dynamic expression of including transcription factors, can also be regulated. these genes during an immune response. This emerging Thus, given their dual functionality to influence knowledge is opening new avenues to better understand chromatin structure and transcription factors, histone epigenetic regulation of inflammatory responses and acetyltransferases and HDACs are at the center of gene providing new molecular targets for either amplifying or regulation. ameliorating immune responses. Histone deacetylases are enzymes that are often Oncogene (2010) 29, 157–173; doi:10.1038/onc.2009.334; recruited by corepressors or multi-protein transcrip- published online 26 October 2009 tional complexes to gene promoters, in which they regulate transcription through chromatin modification Keywords: HDAC; inflammation; cytokine regulation 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; ClassIIincludesHDAC4,5,6,7,9and10;ClassIII consists of members of the family of HDACs; and Class IV is represented by HDAC11, the newest family member discovered (Gao et al.,2002).Although Correspondence: Drs A Villagra or EM Sotomayor or E Seto, histones are a primary substrate for HDACs, several Department of Molecular Oncology, H Lee Moffitt Cancer Center and studies have shown that HDACs also deacetylate non- Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA. histone proteins such as p53, E2F1, RelA, YY1, TFIIE, E-mails: alejandro.villagra@moffitt.org or BCL6 and TFIIF (Glozak et al.,2005).Furthermore, eduardo.sotomayor@moffitt.org or ed.seto@moffitt.org 4These authors contributed equally to this study. although this review will focus on the role of HDACs Received 31 July 2009; revised 5 September 2009; accepted 13 September in gene expression, increasing evidence suggests that 2009; published online 26 October 2009 some HDACs seem to have a wide range of protein Histone deacetylases and immune response A Villagra et al 158 substrates in addition to histones and factors relevant to 2006). For instance, HDAC6 is localized mainly in the transcription. cytoplasm and is uniquely endowed with tubulin Histone deacetylases are the target of several structu- deacetylase activity (Hubbert et al., 2002). This HDAC rally diverse compounds known as HDAC inhibitors is a key regulator of the cytoskeleton, cell migration and (HDIs) (Marks et al., 2000). Of note, these compounds cell–cell interactions (Valenzuela-Ferna´ndez et al., were used as inhibitors long before a clear under- 2008). Moreover, in immune cells, HDAC6 has a role standing of the role of specific HDACs in normal and/or in the formation of the antigen-presenting cell (APC)/ transformed cells began to emerge. These compounds T-cell immune synapse (Serrador et al., 2004). Similarly, can induce cytodifferentiation, cell cycle arrest and HDAC11 is a transcriptional repressor of interleukin apoptosis of transformed cells (Marks et al., 2000; (IL)-10 gene expression in APCs (Villagra et al., 2009) Bolden et al., 2006). Moreover, some HDIs have showed whose expression has been found to be tissue-restricted significant antitumor activity in cancer patients, includ- to the brain, kidney and testis, as well as abnormally ing one compound, SAHA, which has been approved by overexpressed in certain types of leukemias and lym- the Food and Drug Administration for the treatment of phomas (Gao et al., 2002). patients with cutaneous T-cell lymphomas (Mann et al., In this review, we first discuss current knowledge 2007; Marks and Breslow, 2007). In spite of these of the role of specific HDACs in the transcriptional important therapeutic advances, the underlying target(s) regulation of pro- and anti-inflammatory cytokines in and/or mechanism(s) mediating the antitumor activity immune cells. Then, we highlight the emerging regula- exhibited by HDIs in human malignancies remain to be tory role of HDACs in immunological networks fully elucidated. participating in inflammatory responses. Finally, we The HDAC involvement and the therapeutic use of discuss the changes in HDACs that occur in immune HDI are not restricted to cancer, as several studies have cell-derived tumors, such as leukemias and lymphomas, also shown that these enzymes have a role in auto- and how the use of HDIs may potentially tip the balance immune diseases (Pankaj et al., 2008), inflammatory toward effective antitumor responses due to their effects regulation (Blanchard and Chipoy, 2005), central on tumor cells and the host’s immune cells. As there are nervous system disorders (Kazantsev and Thompson, several excellent recent reviews that discuss the topic of 2008) and development (Haberland et al., 2009). Class III HDACs () in immune response and Interestingly, most of the intracellular pathways in- inflammation (Salminen et al., 2008; Finkel et al., 2009; volved in these processes share common intermediaries Natoli, 2009), this review will focus more on the Class I that are regulated by HDACs, suggesting a central role and II enzymes. for these enzymes as regulators of seemingly unrelated physio-pathological conditions. Supporting this con- cept, HDIs have emerged as potential therapeutic tools HDACs and cytokine regulation for the treatment of autoimmune diseases (Mishra et al., 2001; Pankaj et al., 2008), cystic fibrosis (Rubenstein Cytokines are low molecular weight proteins or glyco- and Zeitlin, 1998) muscular dystrophy (Minetti et al., proteins secreted by cells of the immune and other 2006) and regulation of immune tolerance (Tao and systems in response to a variety of extracellular stimuli. Hancock, 2007). Typically produced de novo, these molecules exert their In contrast to the rapidly increasing knowledge of the functions during a very narrow period of time (Cohen role of HDACs in cancer and the use of HDIs in treating and Cohen, 1996). Cytokines can be classified according this and other pathological conditions, still little is to their structural homology, receptor binding capabil- known about the role of specific HDACs in immune ities, immune response and cellular sources. Much cells and the functional consequences of their inhibition evidence points to these molecules playing a central role by HDIs. Although some studies have highlighted the in the regulation of inflammatory responses. Cytokines ability of HDIs to augment inflammatory and antitumor displaying pro- or anti-inflammatory effects have been responses (Blanchard and Chipoy, 2005), others have identified and the kinetics of their production, magni- shown the opposite, that is, HDIs displaying anti- tude and concentration at the site of the immune inflammatory properties and amelioration of graft- reaction are considered as main determinants in shaping versus-host disease and autoimmune disorders (Reddy the initiation, intensity and duration of an immune et al., 2004; Sandra et al., 2005; Glauben et al., 2006). response (Cavaillon, 2001). For instance, IL-12 and 10, Thus, elucidation of the underlying target(s) and cytokines with divergent inflammatory properties, are mechanism(s) involved in these divergent effects of critical in regulating these dynamic changes and main- HDIs remain a challenge given the pan-inhibitory effect taining a delicate balance to prevent autoimmune attack of the compounds currently in use. To complicate things while allowing an efficient immune response against even further, there is accumulating evidence that the role foreign antigens (Moore et al., 2001; Trinchieri, 2003; of specific HDACs in immune cells extends beyond Li and Ra, 2008). histones and now encompasses more complex regulatory Cytokine gene expression can be up- or down- functions that are dependent on the ’s tissue regulated in response to specific stimuli. Such regulation expression profile, cellular compartment distribution, involves several intracellular signaling pathways and stage of cellular differentiation and/or pathophysiologi- occurs very rapidly to affect the cytokine’s transcrip- cal conditions (Glozak et al., 2005; Minucci and Pelicci, tional rate. Of note, a common feature among these

Oncogene Histone deacetylases and immune response A Villagra et al 159 Table 1 Participation of HDACs in the transcriptional regulation of cytokines HDAC Protein target Gene target Comment Reference

HDAC1 Sp1 IL-1 Sp1 recruits HDAC1 to IL-1 promoter Enya et al. (2008) HDAC7, 9 FOXP3 IL-2 HDAC7 and 9 interact with FOXP3 and Li et al. (2007) TIP60 to repress IL-2 expression HDAC1 ZEB1 IL-2 ZEB1 recruits CtBP and HDAC1 to the Wang et al. (2009) IL-2 promoter HDAC1 — IL-12 HDAC1 is recruited to IL-12 promoter Lu et al. (2005) HDAC1 GR IL-5 GR binds to IL-5 promoter and recruits Jee et al. (2005) HDAC1, interfering with GATA3 positive regulation HDAC5 — IL-8 HDAC5 is recruited to IL-8 promoter in Schmeck et al. (2008) cells infected with Legionella pneumophila HDAC2 GR GM-CSF Glucocorticoid receptor recruits HDAC2 Ito et al. (2000) to GM-CSF promoter to antagonize the effect of IL-1b HDAC1, 6, 8 — IFN-b HDAC1 and 8 repress IFN-b expression, Nusinzon and Horvath (2006) and HDAC6 acts as a coactivator to enhance activity HDAC1, 2 Sin3A Ifng Sin3A Recruits HDACs to the Ifng locus Chang et al. (2008) for repress its transcription in Th0 cells HDAC11 — IL-10 HDAC11 is recruited to IL-10 promoter Villagra et al. (2009) to repress transcription HDAC1, 2, 3 — IL-4 HDACs are recruited to IL-4 promoter Valapour et al. (2002) to repress transcription HDAC3 Pro-IL-16 Skp2 HDAC3 binds to pro-IL-16 to repress Skp2 Zhang et al. (2008) expression and subsequently promote cell cycle arrest HDAC1 STAT5, C/EBPb Id-1 STAT5 recruits HDAC1 to Id-1 promoter Xu et al. (2003) for C/EBPb deacetylation, allowing transcriptional activation HDAC3 STAT3 STAT3 target genes HDAC3 enhances STAT3 phosphorylation Togi et al. (2009) HDAC1, 2, 3 STAT3 STAT3 target genes HDACs deacetylate STAT3 to inactivate Yuan et al. (2005) its transcriptional functions HDAC3 STAT1 STAT1 target genes HDAC3 deacetylates STAT1 allowing its Kra¨mer et al. (2009) phosphorylation and nuclear translocation HDAC3, 4, 5 GATA-1 GATA-1 target genes Transcriptional regulation mediated by Watamoto et al. (2003) GATA-1 is diminished in the presence of HDACs HDAC3, 5 GATA-3 GATA-3 target genes Phosphorylated GATA-3 recruits HDAC3 Chen et al. (2006) and HDAC5 to specific DNA regions HDAC3 GATA-2 GATA-2 target genes HDAC3 suppresses the transcriptional Ozawa et al. (2001) potential of GATA-2 HDAC3 NF-kb NF-kb target genes RelA is deacetylated by HDAC3 inducing Chen et al. (2001) its nuclear export

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

different pathways is that they are regulated by matory cytokines. Table 1 summarizes our current molecules promoting the appropriate transcriptional knowledge regarding HDAC regulation of cytokines, microenvironment at the level of the chromatin as well as the target proteins enabling HDAC recruit- substrate. Among these molecules, HDACs influence ment to specific DNA sequences. gene transcription by modifying the cytokine’s promoter regions. The ability of HDACs to also modify non- histone proteins, including transcription factors such as HDACs and regulation of proinflammatory cytokines STAT3, further highlights the important role of these enzymes in cytokine gene regulation (Yuan et al., 2005). Inflammation occurs in response to pathogens, irritants, Indeed, cytokine production by immune cells has damaged cells and other stimuli. This response has been shown to be regulated by changes in the acetyl- been also implicated in the initiation and progression ation status of their promoters (Yao et al., 2005; of cancer (de Visser and Coussens, 2005; Khan and Zhang et al., 2006). Tomasi, 2008). For instance, some malignancies may be The involvement of HDACs in cytokine regulation is prevented by the use of anti-inflammatory drugs not restricted to any specific class or subtype of (Anderson et al., 2006; Rigas, 2007). Inhibition of cytokines. In fact, HDACs appear to participate in the certain proinflammatory cytokines has also been shown transcriptional regulation of both pro- and anti-inflam- to decrease the aggressiveness of some tumor types

Oncogene Histone deacetylases and immune response A Villagra et al 160 (Blanchard and Chipoy, 2005). A better understanding Granulocyte-macrophage colony-stimulating factor of the signal-transduction pathways and transcription (GM-CSF) is produced by immune cells in response to regulators leading to inflammation will likely provide several stimuli. Production of GM-CSF is abrogated important hints toward which pathways and biological following treatment with low concentrations of gluco- processes (for example, apoptosis and/or cell cycle corticoids (Barnes, 2005). HDAC2 has been reported to control) become deregulated in cancer. have a role in this steroid-induced inhibition of GM- For example, IL-8 is a proinflammatory cytokine CSF given: (a) the ability of this HDAC to interact with produced rapidly in response to infection, enabling the glucocorticoid receptor (GR) and (b) the rapid recruitment of granulocytes and macrophages to the recruitment of HDAC2 to the GM-CSF promoter (as injured site (Strieter, 2002). At the molecular level, the observed by chromatin immunoprecipitation) when cells IL-8 promoter is rapidly acetylated by p300, a process are treated with dexamethasone (Ito et al., 2000). that is accompanied by a transient decrease in HDAC1 Furthermore, deacetylation of GR by HDAC2 enables and HDAC5 within its promoter region. At later time interaction between the receptor and nuclear factor points, however, these HDACs are recruited back to the (NF)-kB, promoting its repressor activity upon GM- IL-8 promoter most likely to terminate transcriptional CSF expression (Ito et al., 2006). activity (Schmeck et al., 2008). Interestingly, IL-8 has Finally, IL-5 is a cytokine that has a central role in recently been shown to be overexpressed in several allergic reactions through eosinophil activation. Gluco- malignancies including breast cancer (Freund et al., corticoids are effective drugs for the treatment of several 2003), and its aberrant expression favors angiogenesis as allergic conditions. Recruitment of HDAC1 by GR to well as the growth and invasiveness of malignant cells the IL-5 promoter has been shown to repress transcrip- (Lin et al., 2004). Whether this dysregulation of IL-8 in tion of this gene. Two potential mechanisms have been cancer cells is the result of changes in the expression/ proposed to explain this HDAC1-mediated effect: (a) function of p300, HDAC1 or HDAC5 remains to be recruitment of GR with HDAC1 to the DNA may alter determined. the ability of the transcriptional activator GATA3 to Two other proinflammatory cytokines controlled by bind its DNA target sequence and activate IL-5 gene HDACs are IL-12 and IL-1. IL-12 is regulated at the expression or (b) the HDAC1–GR complex may transcriptional level by histone acetylation. This mod- promote a non-permissive chromatin structure for ification is mediated by p300 to promote gene activa- GATA3 binding (Jee et al., 2005). tion. 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 HDACs and regulation of anti-inflammatory cytokines in various experimental models (Dinarello, 1996). IL-1a gene expression is repressed by a complex consisting of In addition to regulating the expression of proinflam- HDAC1 and Sp1 protein in melanoma cells (Enya et al., matory cytokines, recent evidence has demonstrated 2008). Such modulation of IL-1a expression has been that HDACs have important roles in controlling the proposed to influence the aggressiveness of this malig- transcription of anti-inflammatory cytokines. IL-10 is nancy (Onozaki et al., 1985). one of the best characterized cytokines and is involved Histone deacetylases have also been reported to in tolerance induction and inhibition of inflammatory modulate IL-2, a key cytokine involved in the develop- responses. Recently, we demonstrated that HDAC11 ment, differentiation and homeostasis of T cells. downregulates IL-10 transcription in murine and human Expression of this cytokine is inhibited strongly by APCs by interacting at the chromatin level with the HDAC1 in cooperation with the transcriptional repres- distal region of the promoter (positions 807–1653, sor zinc-finger E-box-binding protein (ZEB)-1 and the relative to the transcription initiation site). Such an corepressor C-terminal-binding protein (CtBP)-2 (Wang effect not only determines the inflammatory status of et al., 2009a). IL-2 expression has been shown to be these cells but also influences priming versus tolerance of deregulated in certain viral and autoimmune diseases as antigen-specific CD4 þ T cells (Villagra et al., 2009). well as in some leukemias (Gesbert et al., 1998). A better Mechanistically, we found that overexpression of understanding of the molecular mechanisms and the HDAC11 in APCs strongly inhibited IL-10 transcrip- consequences of HDAC1-mediated repression of IL-2 tional activity. Chromatin immunoprecipitation analysis gene expression will not only provide important clues to of these cells revealed a lack of histones H3 and H4 the observed IL-2 deregulation but may also unveil acetylation at the IL-10 proximal promoter (positions novel targets for overcoming T-cell anergy, a state of 1–807) that was associated with diminished recruitment unresponsiveness characterized by lack of IL-2 produc- of STAT3 and Sp1. These data most likely reflected the tion in response to cognate antigen (Kametani et al., decreased accessibility of these transcription factors to 2008). FOXP3 is an additional repressor of IL-2 gene less acetylated, and therefore more compact, chromatin. expression that directly associates with the acetyltrans- Importantly, we confirmed the requirement for ferase protein TIP60 (Tat-interacting protein, 60 kDa), HDAC11 enzymatic activity in this process, as over- HDAC7 and HDAC9. HDAC7 and HDAC9 associate expression of an HDAC mutant with a truncated with FOXP3 under different conditions, suggesting that deacetylase domain failed to inhibit IL-10 gene expres- they may have different roles in regulating IL-2 gene sion. Instead, an increase in IL-10 mRNA was observed, expression (Li et al., 2007). perhaps as a result of the HDAC11 mutant acting as a

Oncogene Histone deacetylases and immune response A Villagra et al 161 dominant negative variant and competing with endo- cytokine profile and function during an immune genous HDAC11. Similar enhancement in IL-10 gene response. Some representative cytokines produced by expression was observed when HDAC11-specific short Th1 and Th2 cells are IFNg and IL-4, respectively. In hairpin RNAs were expressed in APCs. In these cells, we addition, the most effective cytokines driving this observed increased H3 and H4 acetylation that was differentiation are IL-12 for Th1 and IL-4 for Th2 accompanied by enhanced recruitment of Sp1 and (Bowen et al., 2008; Aune et al., 2009). Importantly, all STAT3 to the proximal region of the IL-10 promoter. cytokines involved in the differentiation of Th1 and Th2 These changes, together with the absence of negative cells are regulated by HDACs, highlighting their central regulation in the distal promoter mediated by the role in this process. One of the best examples of HDAC transcriptional repressor PU.1 and/or HDAC11, pro- participation in T-cell differentiation is the epigenetic vide a plausible explanation for the enhanced IL-10 gene regulation of the IFNg locus. Most changes in DNA expression observed in HDAC11-deficient APCs. The methylation and acetylation of the IFNg locus occur significance of these findings lies at several levels. First, a within a conserved non-coding sequence >50 kb both physiological role for HDAC11 has been provided. upstream and downstream of the Ifng gene (Zhou et al., Second, HDAC11, by inducing dynamic changes at the 2004), illustrating the importance of the three-dimen- chromatin level, regulates the expression of IL-10 and sional chromatin structure of this locus in its regulation possibly other genes involved in the inflammatory (Aune et al., 2009). response. This effect may explain, at least partially, the Association of HDAC1 and 2 with Sin3A can lead to plasticity of the APC to determine T-cell activation the formation of the transcriptional corepressor com- versus T-cell tolerance. Third, HDAC11 represents a plex Sin3 (Yang and Seto, 2008). The Sin3/HDAC1/2 novel molecular target to potentially influence immune complex is constitutively associated with the IFNg activation versus immune tolerance, a critical decision locus in Th0 cells, thereby repressing the expression with significant implications in cancer immunotherapy, of this cytokine in naive T cells. Following differentia- transplantation and autoimmunity. tion of Th0 cells into Th1 cells, this repression is relieved Class I HDACs have been reported to inhibit IL-4 by displacement of Sin3/HDAC complexes. This process expression in T cells (Valapour et al., 2002). However, is tightly regulated by Th1-related transcriptional re- the mechanisms involved in the recruitment of these gulators such as T-Bet, which promote Sin3/HDAC HDACs to the IL-4 promoter are unknown. Never- complex dissociation (Chang et al., 2008), histone theless, their repressive action on IL-4 expression has acetyltransferase activity, and, ultimately, chromatin raised considerable interest in the potential manipula- acetylation. tion of Class I HDACs for the treatment of asthma The IFNg and T-Bet genes are highly acetylated in (Choi et al., 2005). HDAC1 and 2 have also been Th1, but not Th2 cells, and this hyperacetylation is implicated in the transcriptional control of genes directly associated with their expression. Interestingly, regulated by tumor necrosis factor-a. For instance, when Th2 cells are treated with HDI, the acetylation these HDACs are recruited to the chitinase YKL-40 status of both genes is recovered even though their gene (human cartilage glycoprotein 39) promoter to repress expression is not restored, suggesting a more complex its transcription. Tumor necrosis factor-a in a process mechanism involved in Th1/Th2 differentiation that could be mediated through NF-kB is mainly (Morinobu et al., 2004). A similar mechanism regulates responsible for HDAC recruitment to this promoter the IL-4 gene locus in T-cell differentiation. When naive (Bhat et al., 2008). Of note, YKL-40 has been shown T cells are polarized to the Th2 phenotype, they acquire to induce migration of endothelial cells as well as specific H3K9/14 acetylation (Ansel et al., 2006) that proliferation and protection against inflammatory correlates with enhanced expression of IL-4. In contrast, damage in a variety of cells (Ling and Recklies, 2004). only a slight increase in acetylation of the IL-4 locus is Another anti-inflammatory cytokine, interferon-b observed in Th1 cells (Avni et al., 2002). (IFN-b), is induced in response to some viral infections Interleukin-5 and 13 are two additional cytokines or double-stranded RNA. This induction is dependent expressed in Th2 cells. Both are regulated at the upon the orchestrated action of several transcriptional transcriptional level by GATA3 and GR (Zhang et al., regulators, including different HDACs (Chang et al., 1998; Jee et al., 2005), which have been shown to 2004). Interestingly, although HDAC1 and 8 are interact with HDACs (Chen et al., 2006; Ito et al., 2006). negative regulators of IFN-b expression, HDAC6 acts A current model proposes that GR associates with the as an essential coactivator that induces its transcription IL-5 proximal promoter, where it binds NF-AT and (Nusinzon and Horvath, 2006). This example demon- AP-1. This binding impairs GATA3 recruitment to the strates that different HDACs may regulate the same promoter and activates IL-5 expression. In addition, gene and have opposing roles. 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 HDACs and cytokines involved in T-cell differentiation development and differentiation of T cells (Gesbert et al., 1998). HDAC1 is a strong repressor of IL-2 Naive CD4 T cells differentiate into two types of expression in these cells, and its specific activity on the effector cells, Th1 or Th2. Both subsets differ in their IL-2 promoter is dependent upon the presence

Oncogene Histone deacetylases and immune response A Villagra et al 162 of transcription factors such as ZEB1 and CtBP2 (Wang regulation of target genes (Ihle, 2001). STAT1 is et al., 2009a). Of special interest is the inhibition of IL-2 phosphorylated in response to cytokine and growth in response to bacterial superantigens, a process that is factor stimulation. This event could be counteracted by tightly associated with the ability of HDAC1 to bind the dephosphorylation or CBP acetylation, which promotes IL-2 promoter (Kametani et al., 2008). STAT1 nuclear export (Kra¨mer et al., 2006). Once Finally, HDACs also participate in regulating T-cell acetylated, STAT1 can be deacetylated by HDAC3, as survival. For instance, HDAC7 is a known repressor of well as HDAC1 and 2, thus permitting its phosphoryla- the orphan nuclear receptor Nur77, inhibiting T-cell tion and reactivation (Klampfer et al., 2004; Kra¨mer receptor (TCR)-mediated apoptosis (Dequiedt et al., et al., 2009). Although HDACs promote STAT1 2003). Nur77 is expressed in T cells immediately after activation and nuclear import, their effects on STAT3 TCR activation to promote negative selection is exactly the opposite (Figure 1). STAT3 is acetylated (Woronicz et al., 1994). Overexpression of Nur77 is by p300 in response to cytokine treatment (Paulson associated with apoptosis, whereas its absence inhibits et al., 1999), and this deacetylation is reversed by TCR-mediated cell death (Calnan et al., 1995). The Class I HDACs, leading to impairment of STAT3 functionality of HDAC7 seems to be regulated by dimerization and subsequent transcriptional regulatory reversible phosphorylation, shuttling this deacetylase to activity (Yuan et al., 2005). A recent study has shown the cytoplasm after TCR-dependent phosphorylation HDAC3 interaction with phosphatase 2A (Togi et al., (Parra et al., 2007). 2009), suggesting the existence of a more complicated scaffold of modifying proteins, where phosphorylation and acetylation coordinately regulate each other. Cross-regulation of HDACs and immunological networks STAT5 is also regulated by HDACs. This transcription factor is an activator of the Id-1 gene, a dominant There is special interest in the participation of HDACs negative inhibitor of basic helix-loop-helix transcription in the regulation of STAT and NF-kB signaling and factors such as E2A, HEB and E2-2 (Massari and their relationship to autoimmunity and cancer. HDACs Murre, 2000), and a key regulator of B- and T-cell not only directly activate/repress these pathways but are functions (Engel and Murre, 2001). By recruiting also modulated by these signaling pathways themselves. HDAC1 to the Id-1 promoter to deacetylate C/EBPb, In this section, we will discuss the role of HDACs in the STAT5 can bind DNA and promote Id-1 transcription regulation of these important processes, as well as (Xu et al., 2003). the influence of STAT and NF-kB signaling intermedi- aries in regulating HDAC nuclear localization and degradation. Regulation of HDACs by immunological signaling

Histone deacetylases are regulated many different HDACs in the STAT and NF-kB pathways ways. The magnitude, selectivity and timing of their regulation are dictated by cellular context and the Histone deacetylases associate with multiple proteins to specific combination of events affecting the target form many different constitutive stable complexes, cell. The activities of almost all HDACs are regulated including the Sin3, Mi-2/NuRD, CoREST and by protein–protein interactions and the composition N-CoR/SMRT complexes (Yang and Seto, 2008). of the participating protein complexes. In addition, However, HDACs also associate with proteins in a HDACs are regulated by post-translational modifi- tissue- or stimulus-specific manner. For example, in cation, subcellular localization, transcriptional regula- unstimulated cells, the p50 subunit of NF-kB binds tion, proteolytic processing and availability. HDAC1 to repress the transcription of NF-kB target Class I HDACs are expressed in many different tissues, genes. After activation, the p50/p65 heterodimer trans- whereas Classes II and IV are expressed in a more tissue- locates to the nucleus and displaces p50/HDAC1. This specific manner (de Ruijter et al., 2003). However, mechanism explains the negative role of p50 on regardless of these physiological variations of HDAC transcription (Zhong et al., 2002). A similar mechanism levels, they can be regulated at the transcriptional has been proposed for HDAC3, in which it associates level by different intra- and extracellular signals, with TAB, N-CoR and p50 (Baek et al., 2002). HDAC3 including HDI (Hauser et al., 2002; Bradbury et al., also deacetylates lysine 221 of RelA (p65) in the nucleus, 2005), lipopolysaccharide (LPS) (Aung et al., 2006), promoting NF-kB binding to IkBa and its nuclear phytohemagglutinin (Dangond and Gullans, 1998) and export (Chen et al., 2001). This process terminates cytokines (Bartl et al., 1997). NF-kB activity and recycles protein components to the In Figure 2, three possible mechanisms for regulating cytoplasm for new rounds of activation. In addition, HDACs by immunological pathways and signaling are acetylation of RelA at lysine 310 is required for its illustrated. Subcellular localization and proteolytic transcriptional activity, through an IkBa-independent processing are key elements in many immune signaling mechanism, indicating that acetylation of this protein networks, including the NF-kB (Gopal and Van Dyke, regulates different functions (Chen et al., 2002). 2006) and MAPK pathways (Baek et al., 2002). In Phosphorylation of STATs is required for their addition, expression of several HDACs can be altered activation, nuclear translocation and transcriptional by bacterial components. For example, Legionella

Oncogene Histone deacetylases and immune response A Villagra et al 163

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 Histone deacetylases and immune response A Villagra et al 164 pneumophila infection can increase the expression of (Enya et al., 2008). Because HDAC activity has been HDAC5 but not other HDACs (Schmeck et al., 2008). hypothesized to be reduced by oxidative stress (Barnes LPS stimulation of macrophages affects the expression et al., 2004), it is possible that this biological condition of almost all HDACs, albeit to different magnitudes and may be present in A375-R8 cells, leading to decreased kinetics (Aung et al., 2006). For example, HDAC6, 10 HDAC activity. In addition, HDAC2 activity is reduced and 11 are gradually downregulated up to 8 h after LPS by tyrosine nitration in response to oxidative stress (Ito stimulation with a slight recovery after 24 h. The same et al., 2004; Osoata et al., 2009), and expression of expression pattern is observed for HDAC4, 5 and 7; this HDAC is decreased in smokers (Ito et al., 2001) however, these deacetylases are upregulated 24 h after and patients with chronic obstructive pulmonary disease LPS stimulation. In contrast, HDAC1 is gradually (Ito et al., 2005). upregulated until 8 h, and becomes stabilized by 24 h Finally, pan HDI has been reported to influence the after stimulation. These changes in HDAC expression in expression of some HDACs. Specifically, acute myeloid response to LPS stimulation have prompted the use of cells treated with different HDI show increased selective HDI for controlling the immune response, as HDAC11, 9 and SIRT4 expression (Bradbury et al., these enzymes participate in the expression of pro- and 2005). Another interesting finding is the synergistic anti-inflammatory mediators. action of HDI with phytohemagglutinin for upregulat- Proinflammatory signaling such as those activated by ing HDAC1 and 3 expression in T cells (Dangond and LPS, IL-1b, tumor necrosis factora and IFNg induces Gullans, 1998). HDAC1 ubiquitination and proteosomal degradation. This process involves the same intermediaries as NF-kB signaling (Gopal and Van Dyke, 2006). Once these HDACs in tumors arising from immune cells (hematologic proinflammatory molecules bind to their respective malignancies) receptors, their signals converge onto a common path- way that leads to activation of NF-kB-inducing kinase Epigenetic changes in cancer cells are well-documented and, subsequently, the IkB kinase complex (IKK). IKK phenomena, and there is much interest in understanding induces the phosphorylation and ubiquitination of the exact detailed epigenetic mechanisms in cancer. For IkBa, enabling nuclear import of the NF-kB hetero- example, loss of histone H4-K16 acetylation and histone dimer. A subunit of the IKK complex, IKKb,is H4-K20 trimethylation are distinguishing features in responsible for the ubiquitination of HDAC1 (Gopal many cancers. Importantly, these events appear early et al., 2006). HDACs also regulate the intracellular during tumor initiation (Fraga et al., 2005). An localization of NF-kB and MAPK signaling compo- additional hallmark of tumor cells is DNA methylation. nents. For instance, HDAC3 associates with TAB2 Global hypomethylation and promoter CpG island and N-CoR proteins in a corepressor complex recruited hypermethylation are frequent alterations observed in by the p50 subunit of NF-kB. This complex translocates cancer (Feinberg and Vogelstein, 1983; Esteller, 2002). from nucleus to the cytoplasm upon IL-1 stimulation These findings highlight the major role of epigenetic through MEKK1, which phosphorylates TAB2 to modifiers in cancer. Three major mechanisms have been expose its nuclear export signal (Baek et al., 2002). This identified in the initiation and progression of cancer, mechanism is not restricted to HDAC3 as HDAC1 has namely the activation of silenced genes and/or produc- also been demonstrated to interact with p50 (Zhong tion of fusion proteins, the silencing of tumor suppressor et al., 2002). However, physical interaction between genes and the repression of genes involved in the TAB2 and N-CoR has not been reported for HDAC1 antitumor response. Currently, there is much interest complexes. in understanding the mechanisms governing the regula- Regulation of HDAC availability through intracellu- tion of immune genes in cancer (Khan and Tomasi, lar localization control seems to be a general event. For 2008), such as the involvement of microRNAs (miR- instance, the cellular localization of HDAC7 is regulated NAs) in the altered gene regulation in cancer. Data by reversible phosphorylation. In inactive T cells, indicate that 50% of miRNA genes are located in HDAC7 is located in the nucleus and represses several cancer-associated genomic regions or in fragile sites genes involved in T-cell differentiation, including Nur77 (Sevignani et al., 2006), and that miRNAs are involved (Dequiedt et al., 2003). After TCR activation, the serine/ in the regulation of many immune-related genes, threonine kinase PKD1 phosphorylates HDAC7, thus including major histocompatibility complexes (MHC) promoting its cytoplasmic accumulation. Conversely, and costimulatory molecules (Asirvatham et al., 2009). the myosin phosphatase complex (PP1b/MYPT1) de- This hypothesis proposed by Khan and Tomasi (2008) phosphorylates HDAC7, promoting its nuclear accu- suggests that miRNAs are silencing specific genes by mulation and subsequent inhibition of its target genes binding to nascent RNA transcripts, a process that (Parra et al., 2007). HDAC7 has also been shown to be could be mediated by HDACs as shown at heterochro- cleaved by caspase-8 in T cells tagged by extrinsic matic foci (Tomari and Zamore, 2005). Another apoptotic pathways (Scott et al., 2008). possibility is alteration of the cytokine balance in tumor Modification of HDAC activity is another regulatory cells, and deregulation of the cellular machinery mechanism mediated by cytokines. In this context, responsible for tagging cancer cells for immune recogni- A375-R8 melanoma cell lines expressing IL-1a consti- tion and elimination. For instance, control of MHC tutively have been shown to decrease HDAC1 activity expression can be mediated by cytokines that are

Oncogene Histone deacetylases and immune response A Villagra et al 165 regulated tightly by epigenetic modifiers, including that MHC Classes I and II are repressed by DNA HDACs. 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 Role of HDACs in modulating the immune response expression in cancer cells (Serrano et al., 2001; Karpf against cancer et al., 2004). Direct evidence of HDAC participation in the expression of MHC-I and II has been reported. In T cells recognize antigenic peptides presented on the particular, HDAC1 and 2 repress MHC-II expression in surface of host cells by MHC molecules, of which there human cancer cervical cell lines (Zika et al., 2003), are two types, Class I (MHC-I) and Class II (MHC-II). whereas HDAC1, 2 and 8 repress MHC-I in these cells Both are expressed differentially. Thus, MHC-II is only (Maeda et al., 2000; Magner et al., 2000; Li et al., 2006). found on professional APCs such as dendritic cells, The use of HDI also increases the expression of MHC-I, B cells and macrophages. MHC-I is found on all -II and different APM components and costimulatory nucleated cells and mainly presents peptides of endo- molecules in several types of cancer cells (Khan and genous proteins to CD8 þ cytotoxic T cells. Peptides Tomasi, 2008). In addition, it has been reported that presented by MHC are recognized by T cells, which HDI promotes the expression of MHC Class I-related facilitate the elimination of cells carrying non-self chains A and B, making cancer cells susceptible to peptides derived from endogenous pathogens or elimination by natural killer cells (Skov et al., 2005; mutated proteins, thereby providing an important Zhang et al., 2009). protection against virus infection and tumor mutations. Much effort has been devoted to understanding immune evasion by cancer cells, and how MHC Aberrant expression of HDACs and fusion proteins regulation affects the initiation and progression in cancer of cancer. MHC generation and regulation are key steps to consider. In particular, the mechanisms involved in Alterations in HDAC expression are found in several this process and how intracellular pathways influence types of cancer. For example, HDAC1, 2 and 3 are antigen presentation in cancer cells must be understood. overexpressed in ovarian and endometrial carcinomas Mutations and aberrant expression of MHC-I and (Weichert et al., 2008a), prostate (Weichert et al., 2008b) components of the antigen processing and presentation and renal cancer (Fritzsche et al., 2008). HDAC1 is machinery (APM) have been found in different cancers, overexpressed in cancers of the breast (Zhang et al., including hematological malignancies (Seliger, 2008). 2005), colon (Wilson et al., 2006) and pancreas (Wang For instance, b2-microglobulin has been shown to be et al., 2009b), whereas HDAC3 expression is increased mutated or deleted in colon carcinoma, melanoma and in colon cancer (Wilson et al., 2006). HDAC7 is other tumors (Seliger et al., 2006). Also, the deregulation overexpressed in pancreatic cancer (Ouaı¨ssi et al., of several other components of the MHC-I APM, 2008), and HDAC8 expression is deregulated in occurring at the transcriptional and post-transcriptional neuroblastoma (Oehme et al., 2009). The list of aberrant levels, has been observed (Aptsiauri et al., 2007; Cabrera HDAC expressions in cancer continues to grow and, in et al., 2007; Seliger, 2008). Regulation of MHC-I almost all of them, there is a correlation between high expression seems to be dependent on the aggressiveness expression and poor prognosis. However, there are of tumors and progression of cancer as it has been exceptions. For instance, HDAC6 overexpression shown that lower levels of MHC are related to poor occurs in breast cancer and is associated with a better prognosis in different malignancies (Connor et al., prognosis (Zhang et al., 2004). The silencing of 1993), and metastatic tumors have lower expression of specific HDACs is also associated with reduced aggres- MHC Class I than primary tumors (Cromme et al., siveness, proliferation and cancer survival (Bolden et al., 1994). The loss or downregulation of MHC-I and/or 2006). Also, expression of some Classes II and IV APM components allows tumor cells to evade the HDACs are decreased in neuroblastoma, one of the immune system. Identifying the mechanisms by which most lethal malignant brain tumors (Lucio-Eterovic their expression is regulated will help discover candi- et al., 2008). dates for future treatments against cancer. Only a few hematological malignancies show changes In this context, cytokines have an important role in in HDAC expression. Some subtypes of diffuse large the regulation of MHC and APM components. For B-cell lymphoma exhibit increased HDAC1 expression example, IFNg, tumor necrosis factora, GM-CSF and (Poulsen et al., 2005). In a recent study, HDAC1, 2 and IL-4 are able to upregulate the expression of MHC and 6 expression in diffuse large B-cell lymphoma and APM components (Petersson et al., 1998; Hallermalm peripheral T-cell lymphoma was higher than in normal et al., 2001; Guo et al., 2002). Moreover, IL-10 can lymphoid tissue (Marquard et al., 2009). In this report, repress some APM components (Zeidler et al., 1997). it was suggested that HDAC6 overexpression correlated Thus, HDACs could be participating indirectly in the with a favorable outcome in diffuse large B-cell regulation of these molecules through their capacity to lymphoma patients, whereas the opposite effect was modulate the expression of regulatory cytokines. In observed in peripheral T-cell lymphoma patients. addition, epigenetic modifiers can induce the expression Most irregularities affecting HDACs in hemato- of MHC-I and APM components. Several reports show logical malignancies are associated with the presence

Oncogene 166 Oncogene

Table 2 Examples of HDAC inhibitors in clinical trials

HDACi 1 2 34567891011Combinational drugs Hematological malignancies Clinical trials Possible mechanisms References (NCI, April 2009)

LBH589 xxxxxxxxxxxOngoingstudiesin Cutaneous T-cell lympho- Open: 26 (9 hemato- Decrease chaperone func- Giles et al., 2006; Ellis (Panobinostat) combination with: ma (CTCL). Phase II logical malignancies) tion of Hsp90, leading to et al., 2008; Atadja, Hydroxamate Bortezomib chronic myelogeneous Completed: 12 destabilization and degra- 2009; Frew et al.,2009 (proteosome inh.) leukemia (CML). Phase III (5 hematological dation of growth factors 17-AAG (Hsp90 inh.) malignancies) and their downstream Imatinib (Tyr. Kinase inh.) (AML). Phase II signalings, that is, Lenalidomide Hodgkin’s lymphoma. Bcr-Abl, Flt-3, c-Raf. Phase II Multiple myeloma (MM). itn ectlssadimn response immune and deacetylases Histone Phase I SAHA xxxxxxxxxxxOngoingstudiesin Cutaneous T-cell Lympho- Open: 74 (36 hemato- Induce changes in the Dokmanovic et al., (Vorinostat) combination with: ma (CTCL). Approved logical malignancies) expression of pro- and 2007; Marks and Hydroxamate Bortezomib (proteosome Chronic myelogeneous Completed: 39 antiapoptotic proteins, ac- Breslow, 2007; inh.) leukemia (CML). Phase III (14 hematological tivating the intrinsic Al-Janadi et al., 2008; 17-AAG (Hsp90 inh.) Acute myeloid leukemia malignancies) and/or extrinsic apoptotic Emanuele et al., 2008; Decitadine (AML). Phase II pathways. Possible me- Frew et al.,2009 (hypomethylating agent) Hodgkin’s Lymphoma. chanisms involving cell Etoposide Phase II proliferation and migration. (topoisomerase II inh) Multiple myeloma (MM). Rituximab (CD20 ab) Phase III

Diffuse large B-Cell lym- Villagra A phoma (DLBCL). Phase II Mantle cell lymphoma (MCL). Phase II Chronic lymphocytic leukemia (CLL). Phase II al et PXD101 xxxxxxxxxxxOngoingstudiesin T-Cell Lymphoma. Phase II Open: 9 (6 hemato- Induction of oxidative Feng et al., 2007; (Belinostat) combination with: Acute Myeloid Leukemia logical malignancies) stress and DNA damage in Gimsing et al., 2008; Hydroxamate Bortezomib (AML). Phase II Completed: 8 transformed cells. Enhances Gimsing, 2009; (proteosome inh.) Non-Hodgkin Lymphoma. (4 hematological proapoptotic pathways. Ma et al.,2009 17-AAG (Hsp90 inh.) Phase II malignancies) Azacitidine Multiple Myeloma (MM). (hypomethylating agent) Phase III FK228 (Depsi- x x NA o NA o NA NA NA NA NA Ongoing studies in Cutaneous T-cell lympho- Open: 10 (6 hemato- Activation of intrinsic and/ Furumai et al., 2002; peptide) combination with: ma (CTCL). Phase II logical malignancies) or extrinsic apoptotic Lech-maranda et al., Cyclic peptide Bortezomib Acute Myeloid leukemia Completed: 27 (10 pathways. 2007; Hartlapp et al., (Proteosome inh.) (AML). Phase II hematological Induction of Hsp90 hyper- 2009; Zhou and Decitadine Non-Hodgkin’s malignancies) acetylation. Inhibition of Zhou, 2009 (Hypomethylating agent) Lymphoma. Phase II angiogenesis by downregu- Rituximab (CD20 ab) Multiple myeloma (MM). lation of VEGF expression. Phase II Diffuse large B-Cell lym- phoma (DLBCL). Phase II Mantle cell lymphoma (MCL). Phase II Chronic Lymphocytic leukemia (CLL). Phase II Acute Lymphoblastic lymphoma (ALL). Phase I MGCD0103 xxxooooooxxOngoingstudiesin Acute myeloid leukemia Open: 0 No proposal for possible Bonfils et al., 2008; Benzamide combination with: (AML). Phase II Completed: 7 (7 hema- mechanisms. However, Fournel et al., 2008; Gemcitabine (nucleoside Non-Hodgkin’s tological malignancies) Antiproliferative activity, Tourneau et al.,2008 analog) lymphoma. Phase II cell cycle arrest, and in- 5-azacitidine Hodgkin lymphoma. creased apoptosis has been (hypomethylating agent) Phase II observed in transformed cells. MS-275 x x x o NA o NA o NA o NA Ongoing studies in Acute myeloid leukemia Open: 7 (3 hemato- Generation of reactive Rosato et al., 2003; (SNDX-275) combination with: (AML). Phase II logical malignancies) oxygen species in trans- Hess-Stumpp et al., (Entinostat) Azacitidine Hodgkin lymphoma. Completed: 5 (4 hema- formed cells. 2007; Nishioka et al., Benzamide (hypomethylating agent) Phase II tological malignancies) Enhances proapoptotic 2008 Sargramostin (GM-CSF) pathways. Histone deacetylases and immune response A Villagra et al 167 , of chimeric oncoproteins where aberrant fusion proteins et al. 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) PLZF- RARa (Redner, 2002) and AML1-ETO (Durst and Hiebert, 2008). The consequence of these translocations Kuendgen and Gattermann, 2007; Duenas-Gonzalez 2008 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 downregula- tion of genes involved in specific hematopoietic differ- bition; VEGF, vascular endothelial growth entiation, such as the colony-stimulating factor-1 Enhances extrinsic and intrinsic proapoptotic pathways. Possible mechanisms References (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 lympho- ma, the translocation t(9;14)(p13;q32), which involves the proto-oncogene Pax-5, a transcription factor re- gulating B-cell proliferation and differentiation; follicu- Open: 16 (8 hematological malignancies) Completed: 14 (5 hematological malignancies) (NCI, April 2009) lar 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, re- spectively; Ap12 has antiapoptotic activity, which is also

Acute myeloid leukemia (AML). Phase III Non-Hodgkin’s lymphoma. Phase I Chronic lymphocytic leukemia (CLL). Phase II 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,

trans a transcription factor regulating cell proliferation and growth; anaplastic large T-cell lymphoma, t(2;5)(p23;q35),

binational drugs Hematological malignancies Clinical trials involving Npm/Alk, where Alk is a .

combination with: Decitabine (hypomethylating agent) ATRA (all- retinoic acid) Etoposide (topoisomerase II inh.) 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,

34567891011Com depsipeptide), electrophilic ketones (for example, tri- fluoromethylketone) and miscellaneous compounds (for example, MGCD0103). The inhibitory effect

xxxxxoxxoxNAOngoingstudiesin of HDIs is due to their interaction with the active zinc site in the catalytic pocket of HDACs, as determined by

Continued crystallographic studies of the HDAC8/hydroxamate complex (Somoza et al., 2004). On the basis of their antiproliferative capabilities, HDIs were classified as Valproic Acid Aliphatic acid Table 2 HDACi 1 2 Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating factor; HDAC, histone deacetylase; NA, information not available; o, no inhi factor; x, inhibition. anticancer drugs in erythroleukemic cells (Leder et al.,

Oncogene Histone deacetylases and immune response A Villagra et al 168 1975). However, the molecular mechanism behind their most important HDIs and their use in clinical trials. The functional properties was discovered years later when wide spectrum of hematological malignancies under HDAC inhibition was observed following trichostatin A investigation with HDI, and the promising results treatment of several cell lines, including mammary gland obtained with some of them alone or in combination tumor cells (Yoshida et al., 1990). with other drugs, are notable (Bhalla, 2005; Bishton Histone deacetylase inhibition can result in the et al., 2007; Al-Janadi et al., 2008; Rasheed et al., 2008). induction of apoptosis through extrinsic (for example, However, the use of pan-HDIs lacking specificity does death receptor) and intrinsic (for example, mitochon- not allow for a definitive mechanistic explanation dria) pathways, cell cycle arrest, induction of differ- regarding the action of these compounds on malignan- entiation, antiangiogenic, anti-invasive and immuno- cies. Nevertheless, some investigators propose that their modulatory functions (Bolden et al., 2006; Minucci and effect is mediated mainly by repression of Class I Pelicci, 2006; Dokmanovic et al., 2007). Interestingly, HDACs, as demonstrated by the use of HDIs and most of these effects are seen in transformed cells, specific small interfering RNAs against Class I HDACs whereas normal cells appear to be more resistant to (Glaser et al., 2003; Inoue et al., 2006). According to this HDI (Dokmanovic et al., 2007). The exact mechanisms hypothesis, the use of pan-HDIs could be improved by that allow normal cells to tolerate higher concentrations using the selective compounds that do not target or have of HDI than transformed cells are not completely a lesser effect on Class II HDACs, such as MS-275 understood. However, results suggest that a protective (Class I selective) and MGCD0103 (Classes I, IV and effect of thioredoxin against damage by reactive HDAC10 selective). Without a doubt, the use of oxygen species may be the key (Shao et al., 2004), as selective HDIs will generate more comprehensive cancer cells lack this protein and normal cells with information about the mechanisms of these compounds thioredoxin knockdown accumulate reactive oxygen in treating cancer, providing more accurate effects to be species and display increased cell death (Ungerstedt assigned to simple targets and/or pathways. We must et al., 2005). remember that the use of HDIs for treating cancer not An important number of HDIs have progressed to only affects the proliferation and survival of cancer cells, clinical trials for individual use or in combination with but also other pathways, including immunological other drugs and/or radiation. Table 2 lists some of the 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 Histone deacetylases and immune response A Villagra et al 169 Conclusions different HDACs. Figure 3 depicts a potential model for the action of HDIs on specific genes. This model The pathways and regulators involved in controlling the supports combinatorial steps that ultimately lead to immune response have captured the interest of immu- the repression or activation of particular genes. The nologists and researchers in other areas including cancer specificity of HDIs, along with the expression profile of biology, development and cell cycle control. More HDACs under normal and non-physiological condi- recently, HDACs have been shown to have pivotal roles tions, contributes to the final effects of these in the regulation of immunological pathways. Interest- compounds. Also, indirect effects of HDIs on secondary ingly, the activity of HDACs is not exclusive to the or tertiary proteins that could potentially activate or transcription level; they also regulate non-histone repress target genes should be considered. The combi- proteins, giving a new perspective on the mechanistic natorial model for the action of HDIs must take into action of these enzymes. In this context, HDI are a consideration the cell type, cellular context, physio- potential tool for controlling the immune response pathological condition and HDAC preference. during infection, transplantation, autoimmune diseases and cancer. However, the participation of HDACs in these processes is not completely understood. Moreover, Conflict of interest some information accumulated thus far is contradictory and incomplete. For example, HDAC inhibition can The authors declare no conflict of interest. enhance the immune response in some cases and repress it in others. This duality could be explained by the Acknowledgements differential effect of HDACs on pro- and anti-inflam- matory cytokines, as well as the cellular context. Also, The work in our laboratories was supported by grants to ES considering that HDIs have different preferences for from the National Institutes of Health (NIH), the American particular HDACs, we could argue that the differing Heart Association and the Kaul Foundation, and to EMS outcomes observed by HDIs are due to their effects on from the NIH.

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