Improvement of Malignant Pleural Mesothelioma

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Improvement of Malignant Pleural Mesothelioma Immunotherapy by Epigenetic Modulators Malik Hamaidia1,2,3, Bernard Staumont2, Bernard Duysinx4, Renaud Louis4 and Luc Willems1,2,* 1

AgroBioChem, Agro-Bio Tech, University of Liege, Gembloux, Belgium; 2GIGA, University of Liege, Sart-Tilman, Belgium; 3AgricultureIsLife plateform, University of Liege, Gembloux, Belgium; 4 Pneumology, University hospital of Liege, Sart-Tilman, Belgium Abstract: In the absence of a satisfactory treatment of malignant pleural mesothelioma (MPM), novel therapeutic strategies are urgently needed. Among these, immunotherapy offers a series of advantages such as tumor specificity and good tolerability. Unfortunately, MPM immunotherapy is frequently limited by incomplete cell differentiation or feedback loop regulatory mechanisms. In this review, we describe different components of the innate immune system and discuss strategies to improve MPM immunotherapy by using epigenetic modulators.

Keywords: cancer, epigenetic inhibitors, histone deacetylase, immunotherapy, malignant pleural mesothelioma. 1. INTRODUCTION Malignant mesothelioma is a fatal cancer affecting mesothelial cells from the pleura, pericardium and peritoneum[1]. The development of malignant pleural mesothelioma (MPM) is closely associated with asbestos exposure. Epidemiological studies predict an increase in mesothelioma incidence up to a peak estimated around 2020. Asbestos-exposed subjects present plaques on their parietal and diaphragmatic pleura. The risk of MPM increases with the number of thick pleural plaques (>1cm) as detected by X-ray and computed tomography scanning [2]. MPM development is characterized by a long latency period (20 to 40 years) indicating that multiple molecular events are involved in the transformation of mesothelial cells [2, 3]. DNA damage and chronic inflammation induced by asbestos fibers play a crucial role in tumorigenesis. Histologically, MPM exhibit different phenotypes: epitheloid (50%), sarcomatoid (16%) or biphasic (34%). However, diagnosis of MPM is not trivial and relies on a series of biomarkers: caretinin, Wilms tumor antigen-1 (WT-1), cytokeratin 5/6, mesothelin and vimentin [4-6]. Currently, treatment of MPM is clearly unsatisfactory: neither surgery (extrapleural pleurectomy) nor chemotherapy (cisplatin and pemexetred) significantly improves a mean survival time of 9-12 months [2]. Therefore, other approaches based for example on immunotherapy and epigenetic inhibitors are urgently needed. The idea would be to increase immunogenicity of MPM cells by activating expression of tumor specific proteins.

*Address correspondence to this author at the Department of AgroBioChem, University of Liege, 5030, Gembloux, Belgium; Tel: (+32)4-366-9365,; Fax: (+32)4-366- 4198; E-mail: [email protected] 1568-0266/16 $58.00+.00

2. INFLAMMATION AND MESOTHELIAL CELL TRANSFORMATION Asbestos fibers (amphiboles and serpentines) are heterogeneous minerals whose bio-toxicity depends on the composition and geometry (length, diameter). After inhalation, asbestos is sediments onto the lung parenchyma and migrates to the pleural tissue [3]. The parietal and visceral pleura are attached to the chest wall and to the lung, respectively. The two pleura are composed of a single layer of mesothelial cells interacting with a loose connective tissue and are separated by a thin space that contains pleural fluid. Ciliated epithelial cells and resident macrophages are involved in the clearance of inert material that penetrates the pleura. Retention of asbestos fibers in the lung and in the pleura leads to inflammation and accumulation of interstitial fluid. Thereafter, calcified plaques arise in the parietal pleura [7]. Asbestos fibers passively pass through the visceral pleura and exit via stomata localized on the parietal side. Although the mechanism is still unclear, fiber migration is favored by the drainage of pleural fluids via stomata [8]. Asbestos deposits lead to chronic inflammation characterized by an excessive accumulation of effusions in the pleural space containing immune cells (macrophages, granulocytes and lymphocytes) and inflammatory cytokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-8, vascular endothelial growth factor (VEGF), chemokine ligand 2 (CCL2) and transforming growth factor (TGF)-β1 and TGF-β2 [9, 10] (Fig. 1). Macrophages fail to engulf and digest long asbestos fibers, generating frustrated or incomplete phagocytosis [8]. Direct fiber-induced damages of mesothelial cells leads to an excessive production of reactive oxygen species (ROS) such as superoxide and hydroperoxyl radicals. This oxidative burst involves enzymes such as NADPH oxidase (NOX) that catalyzes the reduction of oxygen to superoxide by using © 2016 Bentham Science Publishers

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Fig. (1). Mechanisms involved in asbestos-related inflammation, mesothelial cell transformation and development of anti-tumor immunity. Asbestos fibers stimulate production of reactive oxygen species (ROS) by mesothelial cells and frustrated macrophages. Concomitantly, asbestos also induces secretion of pro-inflammatory cytokines (IL-6 / TNF-α / IL-1β, VEGF / TGF-α / IL-8 / CCL2 / CCL5 / CX3CL1) and promotes infiltration of leukocytes in the pleural tissue. ROS induce DNA damages and promote mesothelial cell transformation. Transformed cells release tumor associated antigen (TAA) and alarmins captured by resident antigen presenting cells (e.g. DC and macrophages) that differentiate from monocytes. Development of anti-tumor immunity depends on conventional (mDC) maturation and on pro-inflammatory cytokines secreted by classical macrophages (M1). M1 and mDC are able to initiate anti-tumor immunity by presenting captured TAA to naive and memory T-cells in the lymph nodes. mDCs and M1 macrophages drive polarization of CD4 T-cells into antitumor Th1/Th17. Activated cytotoxic T lymphocytes (CTLs) specifically kill tumor cells by using perforin/granzyme. This anti-tumor immunity is inhibited by the development of immature (imDC), semi-mature (smDC) DCs, tumor associated macrophages (TAMs) and regulatory T-lymphocytes (Tregs). These cells induce clonal anergy and deletion of anti-tumor lymphocytes via engagement of Fas/FasL, PD-1/PD-L1/2 and CTLA4/CD86. Moreover, these cells secrete anti-inflammatory cytokines (e.g. IL-10 and TGF-β) involved in the maintenance of an immunosuppressive microenvironment.

NADPH as an electron donor and superoxide dismutase (SOD) that reacts with the superoxide anion to yield hydrogen peroxide. NOX is activated during phagocytosis by association of membrane-bound gp91phox and p22phox proteins that form the flavocytochrome with cytosolic p40phox, p47phox, p67phox and Rac [11]. During frustrated phagocytosis, ROS are released in the extracellular space instead of accumulating inside the early phagosome. Free radicals promote oxidative DNA damage characterized by the generation of 8-hydroxy-2’-desoxyguanosine (8OHdG) and 7.8dihydro-8-oxoguanine (8OdG) that can form hydrogen bonds with deoxycytidine as well as with deoxyadenosine, thereby introducing a mismatch during DNA replication [12]. These genomic alterations can potentially activate oncogenes or alternatively affect tumor suppressor genes leading ultimately to carcinogenesis. Furthermore, ROS favor cell cycle progression by inhibiting p21 and up-regulating proinflammatory cytokines such as TNF-α, IL-6, IL-8, CCL2,

CCL4, IL-1β, CX3CL1 (fractalkine), β-defensin-2 and endothelin-1. Cell survival and proliferation is also promoted by activation of the NALP-3/NLRP-3 inflammasome and signaling through the PI3K-mTOR-AKT and NF-kB pathways [9, 13]. In particular, activation of NF-kB protects mesothelial cells from apoptosis in response to DNA damage by targeting genes that favor cell survival (BCL-xL), proliferation (TNF, IL-1, cyclin D, c-MYC) and angiogenesis (VEGF) [10, 13]. 3. MACROPHAGES AND DENDRITIC CELLS INITIATE ANTI-TUMOR IMMUNITY IN MPM In principle, macrophages and dendritic cells (DCs) are designed to detect tumor-associated antigens (TAA) and initiate anti-tumor immunity. Macrophages and DCs are differentiated from monocytes that are selectively recruited into the pleura. After maturation, these phagocytic cells present

Improvement of Malignant Pleural Mesothelioma Immunotherapy

antigens to naïve and memory T-lymphocytes. The maturation program includes up-regulation of factors involved in cognate interaction with T-lymphocytes (e.g. MHC-I, MHCII, CD80, CD83, CD86). 3.1. Monocyte Subsets Circulating monocytes can be divided into three subsets based on their phenotype: CD14++ CD16- (classical); CD14+CD16++ (non-classical); CD14+CD16+ (intermediate, also called CD14dimCD16+) [14] (Fig. 1). These cells express different chemokine receptors reflecting distinct recruitment properties: CD14++CD16- monocytes are CCR2 high and CCR5/CX3CR1 low while the CD16+ subset is CCR2-negative but expresses high levels of CX3CR1 and CCR5 [15, 16]. Agonists of CCR2 are secreted by macrophages and mesothelial cells after asbestos exposure of the pleura. Classical monocytes are recruited to the tumor site to initiate an anti-tumor immunity and participate to the inflammatory response triggered by pathogens. In contrast, non-classical monocytes attenuate inflammation and are involved either in wound healing or in revascularization. It is known that monocytes initiate their differentiation process during endothelial transmigration. However, selective recruitment and differentiation of monocytes in the pleura is still not well understood [15]. 3.2. Dendritic Cells DCs are professional antigen-presenting cells able to activate both naïve and memory T-lymphocytes. In human, DCs are divided in 2 subsets based on distinct functions: myeloid (or conventional, cDC) and plasmacytoid (pDC) [17]. Conventional DCs differentiate from circulating monocytes in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 (Fig. 1) [17]. DCs are mainly found in tissues in contact with the external environment (e.g. mucosae, dermal tissue) and in lymphoid organs (e.g. thymus, lymph nodes, spleen, Peyer’s patches). Microbial and tumor-derived products induce inflammatory cytokines (e.g. IL-12, IL-6 and IFN- ) involved in activation of macrophages, natural killer (NK), mast cells and granulocytes. Moreover, DCs coordinate and define the nature of the immune function by polarizing naïve T helper lymphocytes (Th) into Th1/Th2 phenotype and promoting activation of tumor specific cytotoxic T lymphocytes (CTLs) (Fig. 1). This ability to control both innate and adaptive immunity requires that DCs undergo a maturation step characterized by an up-regulation of the peptide/MHC complex and costimulatory/adhesion molecules involved in cognate interaction with T-lymphocytes (e.g. CD80, CD83, CD86, CD40L, CD54) [18] . Mature DCs loose innate competence to become a professional antigen-presenting cell (APC). Only fully mature DCs (mDC) are efficient to initiate tumor specific immune response [19]. In fact, mDCs efficiently activate naïve T cells in the lymph node via interaction of the tumor associated peptide/MHC complex with a specific T cell receptor (TCR). A second signal of co-stimulatory molecules, CD80/86 interacting with CD28, is required to achieve T cell engagement. mDCs induce a complete activation of tumor specific CTLs and secrete IFN- , IL-12 and IL-6 that drive polarization of naïve CD4 T-lymphocytes into an anti-tumor phenotype (Th1/Th17) [20] (Fig. 1).

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However, tumor cells produce TAAs that are poorly immunogenic and fail to trigger a complete maturation of DC. In addition, immunosuppressive cytokines generated by tumors and stromal cells stabilize immature and/or semi-mature state of DC (imDC and smDC) [21, 22]. This immature subset is involved in peripheral tolerance by inhibiting effector T cells [19, 23]. Immature DCs are CD80low and express co-stimulatory molecules involved in anergy or apoptosis of effector T cells such as inducible co-stimulator ligand (ICOSL), program death ligand-1/2 (PD-L1/2) and FasL. These cells are also able to produce anti-inflammatory cytokines such as IL-10 and prostaglandin E2 (PGE2) that drive polarization of effector T-cells into inducible regulatory T cells (Tregs) (Fig. 1). The engagement of PD-L1/2 with his ligand program death-1 (PD-1) expressed by effector T cells deliver an inhibitory signal that modulates the balance between T cell activation and tolerance [24, 25]. Anti-tumor T cells secrete IFN- that induces expression of PD-L1/2 by DC, thereby creating a negative feedback loop [26]. This mechanism may thus play a crucial role in tumor immune evasion. 3.3. Plasticity of Macrophages There are two main subsets of macrophages based on their cytokine pattern and phenotype: classical (M1) and alternative (M2) [27]. In fact, macrophages exhibit a broad range of phenotypes between these two extremes. M1 participates in tumor killing by supporting polarization of CD4 T-lymphocytes into anti-tumor Th1 and Th17 whereas tumor associated macrophages (TAMs) that are close to M2 promote survival and proliferation of tumor cells. TAMs originate from myeloid-derived suppressor cells (MDSCs) and CD16+ monocyte precursors [15] (Fig. 1). Polarization of macrophages is controlled by cytokines secreted into the micro-environment. Pro-inflammatory cytokines such as IL1α/β, TNF- α, IFNand IL-12 drive polarization of macrophages into M1 by activating NFkB and Signal Transducers and Activators of Transcription (STAT-1 and STAT4) pathways. In contrast, cytokines such as IL-4, IL-13, IL-5, IL-10, ligands of peroxisome proliferator-activated receptors-γ (PPAR-γ) drive polarization of macrophages into M2 [28]. TAMs and tumor cells secrete anti-inflammatory cytokines (TGF-β, IL-10) promoting development of pro-tumoral regulatory T-cells. Together, these observations thus indicate that DCs and macrophages are major drivers of the anti-tumor response against MPM. This mechanism is finely tuned by the cytokine balance of the tumor microenvironment. 4. MESOTHELIAL TUMOR CELLS EXPRESS ALARMINS, PURINES, NUCLEOTIDES AND TUMOR ASSOCIATED ANTIGENS Compared to their untransformed cell counterparts, MPM tumors express abnormal levels of alarmins, purines, nucleotides and TAAs. 4.1. Alarmins Chronic inflammation in the pleura triggers apoptotic or necrotic death of mesothelial cells. These necrotic cells actively release alarmins, endogenous ligands of TLRs that are


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sensed by pattern recognition receptors expressed by immune cells [29] (Fig. 1). Several cytolosic and nuclear molecules such as heat shock proteins (HSP-90, -70 and -72), high mobility group box-1 (HMGB1), tenascin-C, versican, biglycan, fibrinogen, fibronectin extradomain A and saturated fatty acids are known to be endogenous ligands of the Toll like receptor-4 (TLR4) [30]. By maintaining inflammation in the pleura, alarmin activation of TLRs is a potential driver of MPM. In fact, HMGB1 released by mesothelial cells after asbestos exposure stimulates secretion of TNF-α by macrophages and increases BCL-xL expression thereby promoting survival of transformed mesothelial cells [31, 32]. Components of the extracellular matrix such as tenascin-C are overexpressed by MPM cells and promote the invasive potential by modulating expression of matrix metalloproteinases (MMPs) expressed by macrophages (Fig. 1). The Lipopolysaccharide (LPS) activates Toll-like receptor (TLR) signaling after multimerisation with CD14 and MD-2. LPS induces activation of NFkB, p38, Janus N-terminal kinase (JNK) and interferon regulatory transcription factor 3 (IRF3), providing efficient activation/maturation of antigen presenting cells.

sented by the MHC-I complex constitute a potential T-cell epitope and activate CD8 T-cells. TAA-associated immunogenicity can also induce antibodies directed against the tumor antigens (Fig. 1). By this process, auto-antibodies against mutated P53, Wilms tumor-1 gene product (WT-1) and mesothelin can be generated in MPM patients [37]. These different TAAs are thus potential targets to stimulate immunogenicity against MPM cells [1].

4.2. Purines and Nucleotides

The development of a specific immunotherapy requires the identification of tumor-associated peptides presented by APC and activation of naïve CD8+ T lymphocytes. As indicated previously, MPM tumor cells overexpress different TAAs such as mesothelin and WT-1 [41]. These tumor antigens are also found in other cancers such as lung, ovarian and pancreatic adenocarcinomas but are absent from most normal cells [42]. It is possible to target these tumor cells expressing mesothelin or WT-1 by antigen-specific monoclonal antibodies (e.g. Amatuximab, MORAb-009), conjugated or not with bacterial toxins (e.g. SS1P immunotoxin). Another strategy aims to increase immunogenicity of tumor cells by using live-attenuated Listeria monocytogenes expressing TAA (e.g. CRS-207) [43, 44]. Poorly efficient alone, these antibodies are currently evaluated in clinical trials in combination with standard chemotherapy.

Nucleosides and nucleotides (adenosine, ATP, GTP) are also accumulated in the extracellular pleural space upon chronic inflammation. Adenosine specifically interacts with adenosine receptors (A1, A2A, A2B, and A3) expressed by mesothelial and immune cells. Nucleotides interact with metabotropic (P2Y) and ionotropic (P2X) receptors mediating inflammatory processes such as phagocytosis, cytokine release and chemotaxis [33]. P2X7 receptors induce secretion of IL-1β after interaction with HSP. IL-1β is associated in maintenance of inflammation in the pleura via activation of the NLRP3 inflammasome. Adenosine is produced by degradation of ATP, ADP, and AMP by CD39 and CD73 expressed by infiltrating Tregs. Adenosine induces a potent immunosuppressive effect and accelerates tumor metastasis by inhibiting cytotoxic T-lymphocytes. By antagonizing immunosuppressive cells, enzymes that degrade extracellular adenosine such as adenosine deaminase (ADA) are potential pharmaceutical targets to inhibit tumor growth. 4.3. Tumor Associated Antigens Normal mesothelial cells present self-antigenic peptides to CTLs and NKs generated by the ubiquitin/proteasome pathways through the major histocompatibility complex I (MHC-I). On the other hand, MHC-II molecules expressed mainly by APCs present peptides derived from proteins that are degraded by the endosomal/lysosomal pathways [34]. Normal cells presenting MHC/self-peptides (sp) complex are ignored by NKs and CD8 T-cells. In fact, the MHC-I/sp complex inhibits activation of NKs via engagement of NK inhibitory receptors (KIRs) [35]. In contrast, inhibition of the MHC-I/sp complex in transformed mesothelial cells leads to activation of NKs, NKT and CD8 T-lymphocytes. Tumor cells also abnormally express a variety of proteins involved in cell proliferation, signal transduction, metabolism and adhesion. These tumor-associated proteins lacking an adequate function are poly-ubiquitinated and processed by the cytosolic proteasome [36]. Cleaved proteins are then pre-

5. ADVANCES IN IMMUNOTHERAPY OF MPM Among promising options, immunotherapy aims at modulating the host immune response machinery to target malignant cells [38]. Immunotherapy includes a series of approaches: non-specific improvement of the immune response with cytokines, direct tumor cell killing with engineered antibodies, adoptive transfer of educated cells and vaccination against TAAs. Recently, therapeutic strategies using oncolytic viruses (e.g. measles virus, herpes simplex virus, adenovirus, stomatitis vesicular virus) that preferentially target tumor cells have been developed [39, 40]. 5.1. Immunotherapy to Educate Antitumor Immunity

A second approach is specific adoptive immunotherapy to mimic the development of anti-tumor immunity by using DCs from MPM patients matured ex vivo in presence of autologous tumor lysates [45]. In pioneering experiments, BALB/c mice were injected with bone marrow derived DCs activated with a lysate of AB1 tumor cells. Anti-tumor activity was evaluated upon challenged with AB1 tumor cells. The DC immunotherapy significantly improved survival by generating tumor-specific CD8+ CTLs. The safety of the treatment was then evaluated in a clinical trial with MPM patients receiving adoptive transfer of autologous DC pulsed with tumor lysate (http://clinicaltrials.gov/show/ NCT00280982). Three out of 10 MPM patients showed a partial response after DC-immunotherapy characterized by an increase of peripheral CTLs. A key parameter to activate naïve T-lymphocytes is to obtain mDCs by using cytokines such as IFN- and TNF-α. Other cytokines (IL-2, IL-12, IFN- and GM-CSF) can also be used as adjuvants and improve efficacy of tumor specific CD8+ T cells. In fact, these cytokines increase the activity of antigen presenting cells and drive anti-tumoral polarization of effector CD4 T cells (Th1/Th17) [20, 46].


5.2. Immunotherapy to Antagonize Immunosuppressive Cells An alternate approach of immunotherapy is to antagonize immune cells that inhibit anti-tumoral responses such as TAMs, imDC, smDC and Tregs [47, 48]. Treg cells are able to secrete IL-10 and to convert DCs into imDC that drive anergy of effector T cells [49, 50]. Tregs express cytotoxic T lymphocyte antigen-4 (CTLA-4 or CD152) and program death-1 (PD-1 or CD279), membrane bound and soluble factors involved in immunosuppression. CTLA-4 competes with CD28 to interact with CD80/86 expressed on APCs resulting in a decrease of T cell activation [24, 51]. PD-1 interacts with PD-L1/2 that maintain DC into immature/semi-mature state. Blocking CTLA-4 and PD-1 antagonizes the tolerogenic microenvironment of the tumor site. Antibodies directed against CTLA-4 and the T-cell co-stimulatory receptor 4–1BB increase T-cell infiltration, proliferation, and cytokine production. Consistently, anti-CTLA-4 treatment correlates with a significant decrease of Treg cells [51]. Another way to antagonize the immunosuppressive tumor microenvironment is to use agonists of pattern recognition receptors (PRRs). Ligands of TLR3 (i.e. poly I:C) and TLR7-8 (i.e. immiquimod, R837) indeed enhance production of pro-Th1 cytokines (e.g. IFN- ) by innate cells. In MPM, intra-tumor injection of TLR7 agonists stimulates an effective anti-tumor CTL-dependent immunity in a murine model [52]. 6. HOW EPIGENETIC MODULATORS COULD IMPROVE IMMUNOTHERAPY Immunotherapy is thus definitely a promising approach to treat cancers including MPM. As indicated previously, efficacy of MPM immunotherapy is unfortunately impaired by negative regulatory mechanisms governed for example by M2 macrophage polarization. One potential approach to counter these feedback mechanisms is to use epigenetic modulators that affect expression of key genes involved in cell differentiation or immune tolerance [53, 54]. Gene expression is finely tuned by epigenetic regulation involving reversible modifications of DNA (i.e. cytosine methylation) and histone proteins (e.g. lysine acetylation and methylation) that modulate chromatin architecture and DNA accessibility to transcription factors. In principle, targeting the enzymes that catalyze these modifications could thus improve immunotherapy. These enzymes include DNA methyltrasferases (DNMT1, DNMT3a/b), histone acetyltransferases (HAT), deacetylases (HDAC), methyltransferases (HMT) and demethylases (HDM). Gene inactivation of these enzymes outlined their role in immunotolerance. For example, macrophages lacking expression of histone deacetylase 3 (HDAC3) develop a phenotype similar to IL-4 induced alternative M2 polarization [55]. HDAC3 promotes type I interferon signaling pathways and polarization of macrophages into M1. HDAC7 regulates expression of inflammatory genes in response to TLR stimulation thereby promoting phenotype M1 [56]. DNA hypermethylation contributes to repression of HLA class I antigens, as well as to downregulation of components of the antigen presenting machinery [57]. These epigenetic aberrations may contribute to immune escape in MPM.


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Based on evidence of benefits from combination of epigenetic modulators and classical immunotherapy in some cancer types [58], pharmacological inhibitors targeting enzymes that catalyze epigenetic modifications have been evaluated in MPM. 6.1. Inhibitors of DNMTs A series of DNMT inhibitors have been evaluated for their capacity to increase immunogenicity and immune recognition of MPM cells. The DNA hypomethylating agent 5aza-2'-deoxycytidine (decitabine) induces the expression of cancer/testis antigens in malignant mesothelioma cells [59]. The combination of 5-aza-2'-deoxycytidine with valproate induces cytotoxic T-cell response against mesothelioma [60]. SGI-110 is an improved version of a decitabine linked via a phosphodiester bond to a guanosine [61]. This dinucleotide is resistant to cytidine deaminase efficiently upregulates expression of cancer/testis antigens. Gemcitabine (2′deoxy2′2′-difluorocytidine) induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation [62]. Gemcitabine inhibits DNMTs and reactivates epigenetically silenced genes [63]. Besides increasing immunogenicity of MPM cells, DNMT inhibitors also modulate inflammation controlled by macrophages. By activating suppressor of cytokine signaling (SOCS)-1, 5-aza-2'-deoxycytidine reduces LPS-induced production of TNF-α and IL-6 by macrophages [64]. Although further clinical evaluation is required, pharmacological inhibition of DNA methylation is thus expected to improve MPM immunotherapy. 6.2. HDAC Inhibitors 6.2.1. Sodium Butyrate N-butyrate is a short chain fatty acid able to modulate gene expression and cell differentiation by inhibiting HDAC activity [65]. N-butyrate is produced by intestinal microbes and plays a crucial role in intestinal immune tolerance by modulating functions of immune cells. Nbutyrate exerts anti-inflammatory properties on different immune cell types such as macrophages, dendritic cells and lymphocytes. N-butyrate reduces expression and secretion of pro-inflammatory cytokines by mouse macrophages and dendritic cells in response to TLRs ligands. Furthermore, N-butyrate affects development, maturation and function of human dendritic cells. In fact, butyrate inhibits maturation of dendritic cells by reducing expression of HLA-DR and co-stimulatory molecules (e.g. CD80, CD86, CD40)[66, 67]. The anti-inflammatory effect of butyrate on macrophages is associated with hyperacetylation of histone 3 lysine 9 (H3K9) on NOS2, IL6 and IL12b gene promoters [68]. In addition, butyrate inhibits activated T-lymphocytes expansion by reducing expression and secretion of IL-2 [69]. Furthermore, Nbutyrate induces polarization of CD4+ T cell into regulatory T-cells (Tregs) by promoting hyperacetylation of the Foxp3 locus. Foxp3 is a transcription factor expressed by inducible and natural Tregs that mediate central and peripheral immune tolerance [70, 71].


6.2.2. Trichostatin A Trichostatin A (TSA) is an anti-inflammatory microbial metabolite that inhibits HDAC activity [72, 73]. TSA reduces expression and secretion of pro-inflammatory cytokines (e.g. IL-6, TNF-α, IL-1β, IL-12p40) by murine macrophages in response to TLR agonists [74-76]. TSA induces expression of IL-10, a potent anti-inflammatory cytokine involved in immune tolerance. In addition, TSA disturbs antimicrobial activities of macrophages by reducing phagocytosis and killing of bacteria [77]. TSA inhibits maturation of mouse splenic DCs by reducing expression of costimulatory molecules involved in cognate interaction with T-cells [78]. Moreover, TSA alters the ability of peptidepulsed DCs to induce activation and proliferation of splenic T lymphocytes [79]. TSA reduces production of type I interferon, TNF-α and IL-6 by activated plasmacytoid DCs involved in anti-viral defense [80]. Furthermore, TSA inhibits polarization of naive CD4+ T-lymphocytes into Th17 but promotes differentiation into Tregs with increased immunosuppressive activity [78, 81]. 6.2.3. Suberoylanilide Hydroxamic Acid Suberoylanilide hydroxamic acid (SAHA) is a HDAC inhibitor with potent anti-inflammatory properties. Indeed, SAHA reduces expression of pro-inflammatory cytokines (e.g. IL-6, TNF-α, IL-1α, IFN- ) in mouse macrophages and DCs. SAHA inhibits pro-inflammatory cascades (e.g. NFkB) in response to TLR agonists and impairs activation of murine lymphocytes [74, 76, 82-84]. SAHA also triggers mitochondrial-dependent apoptosis of activated lymphocytes [85]. Although SAHA has been approved for the treatment of cutaneous T cell lymphoma, a trial with 660 MPM patients (NCT00128102) failed to demonstrate its efficacy as single agent to improve overall survival. 6.2.4. Sodium Valproate (VPA) As other HDAC inhibitors, VPA has potent antiinflammatory effects. In fact, VPA reduces secretion of pro-inflammatory cytokines (TNF-α, IFN- , IL-6, IL-17) in a rat experimental model of autoimmune neuritis [86]. VPA drives polarization of naive CD4+ T-lymphocytes into Th2 effector cells and Tregs [86, 87]. VPA reduces expression of pro-inflammatory cytokines (TNF-α, IL12p40, IL-6) and nitric oxide synthase by murine dendritic cells and macrophages in response to LPS [88, 89]. VPA impairs bacterial phagocytosis mediated by mouse bone marrow derived macrophages by inhibiting expression of phagocytic receptors [77]. VPA modulates expression of genes involved in DCs differentiation from myeloid progenitors and monocytes. VPA also inhibits DCs maturation by reducing expression of co-stimulatory molecules [9092]. Despite these negative effects on cell differentiation and anti-inflammatory properties, VPA has antitumor activity in a series of cancers including MPM [93, 94]. VPA augments apoptosis induced by the standard treatment (pemetrexed and cisplatin) in MPM cell lines and in tumor cells from patient’s biopsies. VPA in combination with doxorubicin is well tolerated and is efficient in refractory patients with good performance status [95]. The anti-tumor mechanism is still unknown but may include epigenetic modulation [96], a direct pro-apoptotic effect or immuno-

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modulation such as, for example, up-regulation of NKG2D ligands by γδ T cells [97]. 6.2.5. MS-275 MS-275 is a benzamide that stimulates expression of anti-inflammatory cytokines. In a rat model of experimental autoimmune neuritis, MS-275 favors macrophage polarization towards M2 and production of Tregs [98]. In a model of autoimmune prostatitis, MS-275 attenuates the inflammatory reaction and induces a switch of macrophages from M1 to M2 [99]. Finally, MS-275 alters macrophage response to LPS. Together, these data thus show that different HDAC inhibitors have anti-inflammatory properties despite exhibiting interesting antitumor properties. 6.3. Bromodomain and Extra Terminal Domain (BET) Proteins Acetylated histones are recognized by BET proteins (e.g. Brd4) that mediate a variety of mechanisms such as transcriptional elongation by RNA polymerase II [100]. Bromodomain interacting inhibitors (e.g. I-BET, JQ1) suppress inflammatory gene expression in TLR-stimulated macrophages [101-104]. JQ1 inhibits expression of nitric oxyde synthase-2 and reduces NO production in mice infected with Listeria monocytogenes [105]. In fact, I-BET suppresses a subset of LPS-inducible genes and protects against LPSinduced endotoxic shock in mice. Although the effect of BET inhibitors should be evaluated in MPM, inhibition of TLR-stimulated macrophages does not support their use in immunotherapy. 6.4, Modulators of Histone Methylation Histone methylation is involved either in gene activation (e.g. trimethylation of histone 3 lysine 4) or in silencing (trimethylation of H3K9 and H3K27) depending on location and state of methylation. In particular, methylation of H3K27 is mediated by the EzH2 methyltransferase of the polycomb-repressive complex 2 (PRC-2). Inhibitors of EzH2 (DZNep and GSK 343) impair proliferation and tumorigenicity of MPM cells [106]. Di-methylation of H3K9 is also repressive for gene transcription [107]. Inhibition of G9a, the H3K9 methyltransferase, with BIX01294 represses cell proliferation [108]. Our ongoing experiments indicate that BIX01294 decreases dextran phagocytosis by M2 macrophages (Hamaidia et al., manuscript in preparation). The Jumonji-C domain containing protein, JMJD3 is a histone demethylase specific for H3K27 that catalyses demethylation of H3K27me3 to H3K27me1. In primary mouse macrophages, JMJD3 is TLR-inducible by stimulusregulated transcription factors such as NFkB and is recruited to promoters of LPS-inducible genes [109]. However, gene inactivation shows that JMJD3 deficiency does not impair production of pro-inflammatory cytokines. In fact, JMJD3 is involved in a negative feedback loop of TLR stimulation and is not essential for classical M1 activation. In contrast, JMJD3 is required for M2 macrophage polarization in response to helminth infection and chitin administration [110]. Inhibition of JMJD3 with GSKJ1 could thus decrease alter-


native activation of macrophages and redirect anti-tumor response. In addition to DNA methylation and histone modifications, another mechanism of epigenetic modulation – namely, small and non-coding RNAs - can be targeted pharmacologically. For example, silencing mesothelin by RNA interference decreases viability of mesothelioma cells [111]. Interaction of long non-coding RNAs with the Polycombrepressive complex 2 subunit EzH2 is also a potential target for therapy [112].


Nox

= NADPH oxidase

PDL

= Program death ligand

PGE2

= Prostaglandin E2

PI3K

= Phosphoinositide 3-kinase

PPAR

= Peroxisome proliferator activated receptor

PRC-2

= Polycomb repressive complex-2

PRR

= Pattern recognition receptor

ROS

= Reactive oxygen species

CONCLUSION

SmDC

= Semi-mature dendritic cells

Immunotherapy of mesothelioma currently faces major drawbacks pertaining to tolerance and inadequate cell differentiation. By modulating gene activation and polarization of immune cells, the epigenome dictates the nature of the antitumor immune response. Modulating the epigenome with pharmacological inhibitors could thus break tolerance in MPM. Currently, the major issues relate to the pleiotropic effects of these drugs that affect off target metabolic pathways such as inhibition of non-histone proteins. The development of more selective isoforms may prevent nonspecific targeting and limit toxicity in patients.

SOCS

= Suppressor of cytokine signaling

SOD

= Superoxide dismutase

STAT

= Signal transducers and activators of transcription

TAA

= Tumor associated antigen

TAM

= Tumor associated macrophage

TCR

= T-cell receptor

TGF

= Tumor growth factor

LIST OF ABBREVIATIONS

TLR

= Toll-like receptor

TNF

= Tumor necrotic factor

TSA

= Trichostatin A

VEGF

= Vascular endothelial growth factor

WT-1

= Wilms tumor antigen 1

APC

= Antigen presenting cells

BCL-xL = B-cell lymphoma-extra large

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BET

= Bromodomain and extra terminal domain

CCL2

= CC chemokine ligand 2

CCR2

= CC chemokine receptor 2

CONFLICT OF INTEREST

CTLA

= Cytotoxic T-lymphocyte-associated protein 4

EZH2

= Enhancer of zeste 2

The authors confirm that this article content has no conflict of interest.

Foxp3

= Forkhead box P3

GM-CSF = Granulocyte-macrophage-colony stimulating factor HDAC

= Histone deacetylase

HMGB

= High mobility group box

HSP

= Heat shock protein

ICOSL

= Inducible T-cell co-stimulator ligand

IFN

= Interferon

IL

= Interleukine

imDC

= Immature dentritic cells

IRF

= Interferon regulatory factor

JMJD

= Jumonji-C domain containing protein

LPS

= Lipopolysaccharide

MDSC

= Myeloid derived stem cell

mTOR

= Mammalian target of rapamycin

NALP-3 = NACHT, LRR and PYD domains-containing protein 3 NLRP-3 = NOD-like receptor family, pyrin domain containing 3

ACKNOWLEDGEMENTS MH is a PhD fellow of the "Agricultureislife" program of GxABT. BS and LW are members of the “Fonds National de la Recherche Scientifique” (FNRS). This work is supported by "Agricultureislife", the Synbiofor project of GxABT, the “ULg Fonds Spéciaux pour la Recherche”, the “Action de Recherche Concertée Glyvir” of the “Communauté française de Belgique”, the Télévie, the Interuniversity Attraction Poles (IAP) Program BELVIR initiated by the Belgian Science Policy Office, the Belgian Foundation against Cancer, the Sixth Research Framework Programme of the European Union (project INCA LSHC-CT-2005-018704), the “Neoangio” excellence program and the “Partenariat Public Privé” PPP INCA of the “Direction générale des Technologies, de la Recherche et de l’Energie/DG06” of the Walloon government, the “Centre anticancéreux près ULg” (CAC) and , the “Plan Cancer” of the “Service Public Fédéral”. REFERENCES [1]

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