Therapeutic targeting of myeloid-derived suppressor cells

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Therapeutic targeting of myeloid-derived suppressor cells Stefano Ugel1,2, Federica Delpozzo2, Giacomo Desantis2, Francesca Papalini3, Francesca Simonato2, Nada Sonda4, Serena Zilio1,2 and Vincenzo Bronte4 Myeloid-derived suppressor cells (MDSCs) represent a subset of myeloid cells that expand under pathological conditions, such as cancer development, acute and chronic infections, trauma, bone marrow transplantations, and some autoimmune diseases. MDSCs mediate a negative regulation of the immune response by affecting different T lymphocyte subsets. Potential mechanisms, which underlie this inhibitory activity range from those requiring direct cell-to-cell contact with others, more indirect, and mediated by the modification of the microenvironment. Pharmacological inhibition of MDSC suppressive pathways is a promising strategy to overcome disease-induced immune defects, which might be a key step in enhancing the effectiveness of immune-based therapies. Addresses 1 Department of Oncology and Surgical Science, Via Gattamelata 64, 35128 Padova, Italy 2 Venetian Institute for Molecular Medicine, Via G. Orus 2, 35129 Padova, Italy 3 Laboratory of Tumor Immunology, Scientific-Technological Area, I.N.R.C.A.-I.R.C.C.S., Via Birarelli 8, 60121 Ancona, Italy 4 Istituto Oncologico Veneto - I.R.C.C.S., Via Gattamelata 64, 35128 Padova, Italy Corresponding author: Bronte, Vincenzo ([email protected])

Current Opinion in Pharmacology 2009, 9:470–481 This review comes from a themed issue on Immunomodulation Edited by Francesco di Virgilio and Simon Robson Available online 16th July 2009 1471-4892/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2009.06.014

Introduction Myeloid-derived suppressor cells (MDSCs) are a phenotypically heterogeneous cell population that includes mature myeloid cells, such as granulocytes, monocytes/ macrophages, and dendritic cells (DCs), as well as immature myelo-monocytic precursors [1]. In mice they are defined by the presence of the myeloid-cell lineage differentiation antigens Gr-1 and CD11b. CD11b+Gr1+ cells are normally present only in low numbers in the bone marrow and spleen of healthy mice, while in tumor-bearing mice they accumulate in the spleen, blood, and lymph nodes [1]. Recently, the morphological heterogeneity of MDSCs was further dissected by charCurrent Opinion in Pharmacology 2009, 9:470–481

acterizing the two epitopes recognized by Gr-1-specific antibodies: Ly6G and Ly6C. These epitopes allow for the separation of MDSCs in two different subsets: granulocytic MDSCs (CD11b+Ly6G+Ly6Clow) and monocytic MDSCs (CD11b+Ly6GLy6Chi), which show different distributions and functions under inflammatory related conditions such as cancer or autoimmune diseases [2]. Although the precise relationship between the two subsets is still unknown, recent findings indicate that the monocytic fraction is the major effector of MDSC-dependent immunosuppression likely through a mechanism requiring a functional common a chain of the receptor for the cytokines IL-4 and IL-13 (IL-4-Ra) [3]. In addition, the ability to differentiate into DCs and macrophages in vitro was shown to be restricted to this subset of cells [2]. A connection between tumor-infiltrating macrophages (TAMs) and MDSCs was also recently proposed. According to this hypothesis, circulating monocytic-like MDSCs (CD11b+Gr-1lowF4/80lowIL-4-Ra+) could differentiate into F4/80+ macrophages within the tumor microenvironment and eventually contribute to replenish the pool of TAMs [4]. MDSCs can also interact with TAMs already present within the tumor environment, further promoting their maturation toward alternatively activated macrophages (M2 type). CD31, ER-MP58, low levels of major histocompatibility complex (MHC) class II, and costimulatory molecules such as CD80, which seems to be required for antigen-specific immunosuppression, are additional markers expressed by MDSCs. These immunosuppressive cells have also been described in cancer patients, but their phenotype is less clear than in tumorbearing mice: human MDSCs are usually defined as CD14CD11b+ cells that express the myeloid marker CD33 but neither MHC class II, costimulatory molecules, or other mature myeloid and lymphoid markers [5]. Another cell population associated with negative activity on T lymphocytes in renal cell cancer patients was shown to express the CD15 and CD66 markers, considered to be hallmarks of both mature and immature polymorphonuclear cells [6,7]. MDSC expansion in peripheral lymphoid organs and recruitment to the tumor site depend on tumor-derived soluble factors (TDSFs), which comprise a variety of biologically active compounds, including growth factors, cytokines, and chemokines, structured in a complex pattern of expression and reciprocal cross-talk [8]. MDSCs, recruited during neoplastic growth, sustain tumor progression by providing a favorable microenvirwww.sciencedirect.com

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onment in which transformed cells can proliferate, acquire new mutations, expand, and evade host immunosurveillance; moreover, MDSC subsets can take part in neoangiogenesis [8]. However, the induction of immune dysfunction in the antitumor effectors T lymphocytes is probably the most relevant and commonly cited propriety of MDSCs. The suppressive activity of MDSCs is principally associated with the intracellular metabolism of Larginine, which serves as a substrate for two enzymes: inducible-nitric oxide synthase (iNOS/NOS2), which generates NO, and arginase 1 (ARG1), which converts L-arginine into urea and L-ornithine. The role of these enzymes in MDSC biology has been extensively reviewed elsewhere [9] and here we briefly summarize only some key aspects. ARG1 and NOS2 respond to antithetical stimuli and their regulatory pathways have been traditionally described to be mutually exclusive; however, they can be contemporarily expressed in MDSCs [3]. ARG1-dependent immunoregulatory mechanisms include the decrease in expression of the CD3zchain of T cells [10] and the induction of T cell arrest in G0–G1 cellular phase. By contrast, MDSC-induced production of nitric oxide (NO) by NOS2 is involved in disturbing T cell activation pathways driven by IL-2 receptor. Finally, L-arginine microenvironmental starvation induced by supraphysiological activity of ARG1 could turn NOS2 to change from a physiological NO production to the harmful production of reactive oxygen species (ROS), such as superoxide (O2), which can readily combine with NO to form reactive nitrogen species (RNS), such as peroxynitrites, able to alter intracellular signaling and induce apoptosis in T lymphocytes [11]. In MDSCs, peroxynitrites can also be generated by the combined activity of phagocytic oxidase together with NOS, which not necessarily has to be NOS2 [12]. The role of MDSC-derived ROS and RNS is complex because these molecules cause DNA damage in tumor microenvironment, prevent maturation of immature MDSCs into fully functional DCs, and also impair the function of tumor-specific T cells by nitrating the T cell receptor (TCR) and altering its binding to the MHC class I molecule and peptide complex [12]. Since MDSCs fuel one of the main immunosuppressive circuits in cancer, several pharmacological approaches, which involve either MDSC elimination or modulation of their functions, are currently being explored in tumorbearing hosts. Moreover, these novel approaches could be potentially translated to the therapy of other diseases in which MDSCs can play a pathogenic role, such as immunosuppression/immune deviation associated with chronic infections. For simplicity, we can divide these MDSC inhibitors in four classes according to their ability to control: firstly, MDSC differentiation into mature cells; secondly, MDSC maturation from precursors; thirdly, MDSC accumulation in lymphoid organs; and fourthly, MDSC function. www.sciencedirect.com

Drugs forcing MDSC differentiation into mature cells A promising approach in cancer immunotherapy is targeting MDSCs to promote their differentiation into mature myeloid cells that no longer possess suppressive activity [1]. The rationale behind this approach is the idea that the conversion of MDSCs could improve antitumor immune responses and enhance the effect of cancer vaccines or adoptively transferred tumor-specific T lymphocytes. Vitamin A is known to favor the differentiation of myeloid progenitors into DCs and macrophages, and its deficiency causes an expansion of immature CD11b+/Gr-1+ myeloid cells in the blood. In addition, vitamin A deficiency may compromise adaptive immune responses depending on myeloid DC antigen presentation. Two types of nuclear hormone receptors, RAR and RXR, mediate retinoid biologic activities. Both RAR and RXR bind retinoids and analogs through the ligand-binding domain [13]. These receptors form heterodimers, RAR/ RXR, that modulate the frequency of transcription initiation of target genes after binding to retinoic acid response elements (RARE) in their promoter regions [14]. The recent generation of highly specific pan-RAR antagonists makes it possible to assess the specific role of RAR signaling branches, allowing for a distinction between RAR-dependent and RXR-dependent pathways. Administration of a synthetic antagonist of the three RAR isotypes (a, b, g), which does not interact with RXRs, elicited an accumulation of granulocytes in different hematopoietic compartments of mice, including the bone marrow [15]. These data suggest an essential role for RAR signaling in regulating the number of granulocytic precursors in vivo. It is not surprising, therefore, that alltrans-retinoic acid (ATRA), a derivate of vitamin A, has been studied for its potential role in forcing the differentiation of MDSCs into mature granulocytes. Nefedova and colleagues showed that ATRA upregulates glutathione synthase (GSS), one of the enzymes required for glutathione synthesis, therefore determining the accumulation of glutathione in myeloid cells. This was observed both in mice and in cancer patients [16]. ATRA was shown to induce MDSC differentiation by neutralizing high ROS production, that is considered essential for maintaining the immature state of MDSCs. Further experiments revealed that ATRA regulates GSS expression not by directly binding its promoter but, primarily, through the activation of extracellular signal-regulated kinase 1/2 [16]. Physiologic concentrations of ATRA (1 mM) in combination with GM-CSF (20 ng/ml) induced in vitro differentiation of MDSC from tumor-bearing mice into CD11c+/MHC class II+ myeloid DCs [17]. ATRA administration did not directly inhibit the growth of solid tumors but resulted in increased proportion of DCs, Current Opinion in Pharmacology 2009, 9:470–481

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macrophages, and granulocytes among adoptively transferred CD11b+/Gr-1+ cells isolated from the spleen of tumor-bearing mice. These adoptive transfer experiments, thus, indicate that ATRA forces in vivo MDSC differentiation in DCs, macrophages, and granulocytes. The decreased presence of MDSCs in tumor-bearing mice also enhanced CD4+ and CD8+ T cell-dependent, tumor-specific immune responses. Furthermore, the combination of ATRA with two different cancer vaccines, in two different tumor models, significantly prolonged the antitumor effect of the treatment, suggesting ATRA as a possible candidate for enhancing the effectiveness of active immunotherapy of cancer [17]. All previously mentioned studies were performed in mice, but recently Mirza et al. addressed the effect of ATRA on MDSC in cancer patients [18]. They treated 18 patients with a metastatic renal cell carcinoma with ATRA followed by subcutaneous IL-2 injection. Although ATRA was well tolerated, it had a rather heterogeneous absorption and clearance among individuals. ATRA did not have any effect on either phenotype or function of DCs and MDSCs at concentrations below 150 ng/ml. In fact, a substantial decrease in the number of MDSCs in the peripheral blood and improved antigenspecific T-cell response was observed only in patients with high plasma concentration of ATRA (>150 ng/ml). Interestingly, during IL-2 treatment, the ATRA effect was completely eliminated. To clarify the role of IL-2, 15 patients with metastatic RCC received intravenous IL-2 alone. In this group, IL-2 significantly reduced the number and function of DCs as well as T-cell function, suggesting that ATRA probably should not be combined with IL-2 in cancer vaccines if the purpose is to improve the host immune responses. A possible explanation could be that the accumulation of MDSC and T regulatory (Treg) cell population in patients treated with IL-2 could limit the clinical efficacy of this therapy [18]. Some hematological malignancies, such as the acute myelogenous leukemias (AMLs), are generally refractory to retinoic-induced differentiation. The majority of acute promyeloid leukemia cases are characterized by the expression of PML-RARa, an aberrant form of RARa. The activity and stability of nuclear receptors, including PML-RARa, are modulated by various signals, such as phosphorylation events. The proteolytic degradation of these factors via the proteasome-dependent pathway is an emerging control system for the activity of retinoic receptor complexes. In AMLs the peptidyl-propyl-isomerase Pin1, which isomerizes the Ser(Thr)-Pro bond from cis to trans altering the conformation of target proteins, is highly expressed. Pin1 is able to induce the degradation of the nuclear receptor (RAR, receptor for ATRA) via the proteasome-dependent pathway. Suppression of Pin1 activity stabilizes the RAR and PML-RARa, resulting in increased sensitivity to the differentiating and antiCurrent Opinion in Pharmacology 2009, 9:470–481

proliferative activities of ATRA [19]. These findings suggest that Pin1 might represent an important target for strategies aimed at increasing the therapeutic index of retinoids. Other MDSC differentiating molecules have been exploited experimentally. Patients with head and neck squamous cell carcinoma (HNSCC) were orally treated with dosage of 60 mg/day with 25-hydroxyvitamin D3. Assays evaluating immune reactivity among peripheral blood leukocytes demonstrated that the treatment of HNSCC patients with Vitamin D3 reduced the number of immune suppressive CD34+cells, increased HLA-DR expression in circulating PBMC, increased plasma IL-12 and IFN-g levels, and improved T-cell blastogenesis in vitro [20]. Vitamin D3 may thus represent another agent with the potential to decrease MDSC numbers in patients with cancer, as well as to promote myeloid-cell differentiation.

Drugs inhibiting MDSC maturation from precursors Another attractive strategy is based on neutralizing the factors that are involved in MDSC expansion from the hematopoietic precursors. Although promising, this task is made complex by the plethora of TDSFs involved in MDSC expansion and recruitment, which includes: prostaglandins; growth factors like granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), stem-cell factor (SCF), vascular endothelial growth factor (VEGF); cytokines like transforming growth factor-b (TGF-b), IL-1b, IL-6, IL-10, IL-12, IL-13; and chemokines, like CC-chemokine ligand 2 (CCL2), CXC-chemokine ligand 5 and CXCchemokine ligand 12 (CXCL2 and CXCL12). Some of these factors, however, trigger a common intracellular pathway in MDSCs that involves the signal transducer and activator of transcription (STAT) family of transcription factors. In mammalian cells, seven different isoforms of STAT proteins have been identified, encoded by distinct genes but all constituted by a DNA-binding domain in the N-terminus and a Srchomology 2 (SH2) domain in the C-terminus. The SH2 domain is required for STAT activation and dimerization. The dimers are the active forms of the STAT proteins. In normal cells, STAT protein activation is tightly regulated by numerous mechanisms that is dephosphorylation of either the receptor complexes or nuclear STAT dimers by protein tyrosine phosphatase (PTPases), and/or activity of protein inhibitors of activated STAT (PIAS), and/or feedback inhibition of the Jak/STAT pathway by the suppressor of cytokine signaling (SOCS) proteins [21]. Among the members of this family, STAT3 is well recognized as a key regulator of MDSC biology. MDSCs are characterized by a persistent STAT3 activation www.sciencedirect.com

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induced by various alterations in tumor microenvironment, including oncogenic activation of receptor tyrosine kinases and release of IL-6, VEGF, and IL-10 [22,23]. The consequences of STAT3 constitutive activation is the upregulation of the numerous STAT3-dependent genes, among which there are antiapoptotic (BCL-XL), proproliferative (survivin, cyclin D1/D2), and proangiogenic proteins (MMP2, MMP9, and HIF1a) [23]. This persistent STAT3 activation also contributes to the increased production of ROS by MDSCs [1]. STAT3 is thus a rather interesting target for the development of novel adjuvants in cancer immunotherapy. Importantly, activated STAT3 inhibitors could have little or no effect on normal cells, where, as described above, its activation is highly regulated. Several molecules have been already tested as STAT3 inhibitors. In particular, many studies exploited either inhibition of STAT3 recruitment to activated tyrosine kinases or prevention of dimer formation and STAT3-DNA binding. Chemically, STAT3 inhibitors can be classified into four different groups: peptides, peptidomimetics, small molecules, and platinum complexes. Peptides are small amino acid sequences that, binding to SH2 domain of STAT3, reduce its interaction with activated receptors, thus blocking the first steps of STAT3 pathway signaling. However, despite showing elevated STAT3 inhibition in vitro, peptides have limited cell permeability, making in vivo therapy possible only at relevant dosages. For this reason, peptidomimetic inhibitors represent a more reasonable alternative, showing both higher cell permeability and resistance to protease digestion [24]. Alternatively, STAT3 inhibitors have been identified through the in silico screening of chemical libraries. Structure-based virtual screening of more than 400 000 compounds from the National Cancer Institute, Merck, Sigma–Aldrich and Ryan databases, allowed for the identification of two small molecules, STA-21 and S31201, which can block the STAT3 pathway with high selectivity showing little toxicity in normal cells [25]. In a recent work, a cell line expressing a luciferase reporter gene under the control of a STAT3-promoter was used to screen for the 1120 bio-compounds of the Prestwick Library. This screening identified nifuroxazide as a potent STAT3 inhibitor in multiple myeloma cells by blocking STAT3-phosphorylation through inhibition of the Jak family kinases, Jak2 and Tyk2 [26]. The fourth group of STAT3 inhibitors is the platinum complexes. These complexes possess anticancer action by cross-linking DNA, but recent studies have shown that some of them can also inhibit STAT signaling, mainly the STAT3 pathway, with selective and strong effects. Moreover, at least for one of these compounds, CPA-7, the antitumor response without a direct action on tumor cells was proven. Actually, an indirect activity on tumor immuwww.sciencedirect.com

nity was postulated. In particular, CPA-7 treatment reduced phosphorylated STAT3 levels in tumor-infiltrating DCs and, additionally, activated NK antitumor response. Of note, this transient molecular STAT3 modulation in immune cells did not induce the development of autoimmune diseases, as reported previously for complete STAT3 gene-ablation [27], supporting the feasibility as a therapeutic agent. A selective inhibitor of the Jak2/STAT3 pathway, JSI-124 (curcubitacin I), was also capable of increasing immune responses against tumors [28]. This molecule markedly reduced the number of CD11b+Gr-1+ immature myeloid cells in the tumor microenvironment, both by increasing their apoptosis (up to 50% in a colon carcinoma model) and by promoting their differentiation into mature cells. The exact mechanism of action of JSI-124 is not completely known. It was hypothesized that it could increase dephosphorylation of STAT3 through activation of phosphatases, or via PIAS and SOCS protein activation. One important aspect to consider is that constitutive ablation of the STAT3 gene or continuous drug administration cannot be considered for therapeutic purposes, since the STAT3 pathway is necessary for normal hematological differentiation and function. By contrast, intermittent use of pharmacological drugs, with limited halflife, could guarantee a very efficient antitumor strategy, without overt interference with normal hematological development and activity of the immune system. To date, however, none of the many STAT3 inhibitors has been used in clinical trials. Another category of drugs comprises known molecules already used in the clinic which might target STAT3 in addition to other factors. Sunitinib, for example, is used for the treatment of several tumor types for its alleged antiangiogenic properties. It was recently reported that Sunitinib, a tyrosine kinase inhibitor, acts also by inhibiting the STAT3 pathway in renal carcinoma-associated MDSCs [29]. Tyrosine kinases catalyze the transfer of the g-phosphate group from adenosine triphosphate to target proteins. They can be classified as either receptor protein kinases or nonreceptor protein kinases. The receptor tyrosine kinases are membrane-spanning cell surface proteins that play critical roles in the transduction of extracellular signals to the cytoplasm [30]. There are approximately 60 receptor tyrosine kinases identified so far, which are characterized by immunoglobulin-like sequences in their amino terminal extracellular domains, a lipophilic transmembrane segment, and an intracellular carboxyl-terminal domain that includes the tyrosine kinase catalytic site. Nonreceptor tyrosine kinases, by contrast, relay intracellular signals. Ligand binding induces dimerization of these receptor tyrosine kinases, resulting in the autophosphorylation of their cytoplasmatic domains and activation of tyrosine kinase activity. Current Opinion in Pharmacology 2009, 9:470–481

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Multiple cytoplasmatic signaling pathways, including the Ras/Raf mitogen-activated protein kinase, the STAT3 and the protein kinase C pathway, in addition to scaffolding proteins may then be activated. Several tyrosine kinase inhibitors were found to have effective antitumor activity and were approved for, or are already in clinical trials. These include imantinib (STI 751; Gleevec), which inhibits the nonreceptor tyrosine kinases BCR-ABL and KIT, as well as receptor tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR) (ErbB/HER) family members, such as gefinitib (Iressa) or erlotinib (OSI-774; Tarceva), platelet-derived growth factor receptors (PDGFR), such as leflunomide (SU101), and vascular endothelial growth factor receptors (VEGFR) such as samaxinib (SU5416), vatalanib (PTK787/ZK222585), and sorafenib (BAY 439006) [31]. Sunitinib (SU11248; sutent) is a broad-spectrum inhibitor that blocks the signaling through the VEGFR, as well as the PDGFR, the stem cell factor receptor (c-Kit), Flt3, and the CSF-1 receptor. Phase I trials have reported tumor regressions together with antiangiogenic activity, and a phase II studies in patients with metastatic kidney cancer found that 33% of patients had a partial response, while 37% had a stable disease for more than three months after therapy [32,33]. Recently, a significant reduction in circulating MDSCs in metastatic renal cell carcinoma (mRCC) patients after sunitinib therapy was reported. Moreover, peripheral blood leukocytes analyzed from metastatic cancer patients revealed significant differences in marker profiles between samples collected before and after treatment with sunitinib. In addition, it was shown that sunitinib treatment promoted CD1c+ DC expansion, suggesting an immune-modulatory effect of sunitinib in cancer patients; however, the effect of sunitinib on CD14+ myeloid suppressor cells in mRCC patients was not statistically significant after four weeks of treatment [34]. More importantly, Finke and colleagues reported a significant increase in Th1 response and a diminished Th2 cytokine expression after four weeks of sunitinib treatment, together with an overall decrease in Treg population [35], which is similar to a treatment with the anti-c-Kit monoclonal antibody [36]. It is possible that the reduction of Treg cells by sunitinib is mediated by a downregulation of MDSCs. Furthermore, Sunitinib administration was able to improve tumor-infiltrating lymphocyte (TIL) function, reducing the tumor microenvironment levels of IL-10, Foxp3, PD-1, CTLA4, and BAFF (B lymphocyte-activating factor belonging to tumor necrosis factor superfamily) [36]. Finally, it was demonstrated that Sunitinib inhibited Src/STAT3 activity in tumor-associated MDSCs, reducing expression of several STAT3-regulated angiogenic genes in MDSCs [37]. These findings support the hypothesis, as further discussed below, that MDSC subsets are also involved in promoting tumor stroma remodeling and angiogenesis. Current Opinion in Pharmacology 2009, 9:470–481

Antiangiogenic therapy based on the modulation of VEGF has also been considered as a possible approach to manipulate MDSC expansion. VEGF is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth, and reproductive functions, and it has been implicated in pathological angiogenesis associated with tumor development [38]. VEGF also affects bone marrow-derived cells, promoting monocyte chemotaxis and inducing colony formation by subsets of granulocytemacrophage progenitor cells. VEGF delivery to adult mice inhibited DC development, increased production of B cells and led to the generation of immature MDSCs [39]. In situ hybridization studies have shown that VEGF mRNA is upregulated in many human tumors. In 1993, Kim et al. reported that monoclonal antibodies (mAbs) to VEGF exerted a potent inhibitory effect on the growth of several tumor cell lines xenotransplanted in nude mice [40]. Subsequently, many other tumor cell lines were found to be inhibited in vivo by anti-VEGF mAb treatment. From these seminal observations, therapies have been developed to inhibit VEGF signaling pathways by targeting either the ligands (VEGF-A) or the receptors (VEGFR-1 and VEGFR-2) [38]. VEGF-A blockade using the humanized anti-VEGF-A mAb bevacizumab (Avastin) was found to significantly inhibit angiogenesis and growth of human tumor xenografts [41]. Avastin was the first antiangiogenic agent approved by the Food and Drug Administration for the treatment of metastatic colorectal cancer and nonsmall cell lung cancer in combination with cytotoxic chemotherapy. Administration of this antibody was shown to cause also a decrease in the pool of a MDSC subset (CD11b+VEGFR1+ cells) in the peripheral blood of RCC patients [42]. However, tumors can grow in the absence of VEGF. Ferrara and colleagues recently suggested that the priming and recruitment of CD11b+Gr-1+ cells might represent a cellular mechanism underlying the resistance that some tumors show in response to anti-VEGF mAb treatment. They demonstrated that anti-VEGF therapy, in combination with a mAb that targets CD11b+Gr-1+ myeloid cells, partially overcomes tumor refractoriness [41]. The proangiogenic activity of CD11b+Gr-1+ cells was found to depend, almost entirely, on the production of the ortholog of the secreted protein Bv8 [43]. Bv8, also called prokineticin-2, belongs to a larger class of peptides that is defined by a five disulfide bridge motif, called a colipase fold. Both Bv8 and its structurally related protein, endocrine gland vascular endothelial growth factor (EG-VEGF), bind two highly homologous Gprotein-coupled receptors termed EG-VEGRF/PKR-1 and EG-VEGFR/PKR-2. EG-VEGF and Bv8 were characterized as mitogens selective for specific endothelial cell types [44]. Furthermore, Bv8 or EG-VEGF induces hematopoietic cell mobilization in vivo and www.sciencedirect.com

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stimulate the production of granulocytic and monocytic colonies in vitro [45]. These activities, combined with the expression of Bv8 (but not of EG-VEGF) in the bone marrow, make Bv8 an interesting candidate for inflammatory cell-dependent angiogenesis [43]. Shojaei et al. showed that Bv8 is upregulated in bone marrow mononuclear cells (BMMNCs) in immunodeficient mice after tumor implantation; in particular, BV8 was highly expressed in CD11b+Gr-1+ fraction compared to CD11bGr-1 cells [41,43]. In vitro experiments established that Bv8 expression was dramatically upregulated by G-CSF whereas GM-CSF had no effect, suggesting a high degree of specificity in Bv8 regulation. Furthermore, in vivo administration of recombinant GCSF to BALB/c mice resulted in a time-dependent and dose-dependent increase in Bv8 levels in the BMMNCs and in the serum, which coincided with an increase in peripheral blood neutrophils [41,43]. Further in vivo studies, performed to confirm the role of G-CSF in regulating Bv8 expression, showed that Bv8 protein levels are significantly reduced in the bone marrow of nontumor-bearing mice treated with anti-G-CSF as compared to controls. In addition, anti-G-CSF antibody virtually abolishes the peak in Bv8 protein levels occurring in the bone marrow shortly after tumor implantation. It was also shown that anti-Bv8 treatment, shortly after tumor cell inoculation, affected the growth of several tumor cell lines, resulting in a significant decrease in tumor volume and weight compared to control animals [41,43]. Interestingly, anti-Bv8 mAb treatment reduced the number of tumor-associated CD11b+Gr-1+ cells, suggesting that Bv8 might also regulate mobilization and, potentially, homing of CD11b+Gr-1+ cells to the tumor site [43]. Another possible target for a therapeutic approach to block MDSC expansion is the matrix metalloproteinase-9 (MMP-9). The matrix metalloproteinases (MMPs) are a family of closely related, zinc-dependent proteolytic enzymes. They are able to degrade all the components of the extracellular matrix and, as such, are involved in a number of physiological and pathological processes. The extracellular matrix is the principal barrier to tumor growth and spread, and there is evidence that MMPs play a role in the processes of tumor spreading and metastasis. MMP-9 is a stromal factor, highly expressed in the tumor area and increased levels of pro-MMP-9 were found in the sera of tumor-bearing mice. MMP9 regulated the mobilization of hematopoietic stem cells from the bone marrow niche and MDSC expansion by solubilizing the membrane form of c-KitL [46] and making VEGF available. MMP-inhibitors can block the MMP-9 expression at the tumor site, halting this loop and normalizing hematopoiesis, thus reducing both myeloid support to tumor stroma and MDSC generation. www.sciencedirect.com

However, the attempts to block MMPs directly in order to prevent tumor progression have failed in phase III clinical trials [47]. Amino-biphosphonates represent an interesting alternative since they have shown excellent results in terms of safety and tolerance. Melani et al. [48] used two different amino-biphosphonates, pamidronate, and zoledronate. These molecules interfere with protein prenylation and with the conversion of mevalonate to geranylgeranylpyrophosphate necessary for cholesterol synthesis. Amino-biphosphonate treatment reduced both myelopoiesis and tumor stroma formation, enhancing tumor necrosis. Within the tumor stroma, the infiltration of inflammatory CD11b+Gr-1+F4/80+ cells was significantly reduced by amino-biphosphonate treatment [48]. These studies also confirmed that a prolonged treatment with amino-biphosphonates had no detectable toxicity in mice. In cancer patients a single dose of pamidronate and zoledronate decreased serum levels of VEGF, TGF-b, and MMP-2. This suggests multifactorial effects which might include a blockade of osteoclast bone resorption, CXCR-4-guided metastastic spreading, inhibition of MMP-9, as well as activation of gd T lymphocytes [49]. Future experiments are needed to dose pamidronate and zoledronate for clinical-safe regimens and evaluate the effective value of the preclinical studies.

Drugs reducing MDSC accumulation in peripheral organs Some strategies used in the past, such as the in vivo administration of mAbs against Gr-1, aimed at depleting Gr-1+ MDSCs, gave interesting results in restoring T cell antitumor activity, both in terms of general reduction of tumor progression and in terms of prevention of relapse [50,51]. Unfortunately Gr-1, as other nonspecific markers of mouse and human MDSCs, is also expressed on mature granulocytes, implying the possibility that tumor-bearing hosts treated with depleting antibodies might undergo opportunistic infections. Moreover, a strong rebound accumulation of CD11b+Gr-1+ MDSCs occurs rapidly when the concentration of anti-Gr-1 mAbs drops in the serum or when an antibody response to the rat anti-Gr-1 mAb develops in the mouse (unpublished data), making use of this approach rather unpredictable. Therefore, either chemotherapy or host irradiation has been used to eliminate MDSCs. In fact, the administration of chemotherapy drugs, such as gemcitabine, to mice bearing large tumors, reduced the number of splenic MDSCs, whereas the number of CD4+ T cells, CD8+ T cells, NK cells, macrophages, or B cells was apparently not affected [52]. Gemcitabine is a nucleoside analog of cytidine in which the hydrogen atoms on the 20 carbons of deoxycytidine are replaced by fluorine atoms. After the incorporation of this molecule into the DNA, only one other nucleotide can be added to the DNA strand causing masked chain termination [53]. Nucleoside analog accumulation inhibits the activity of ribonucleotide reductase, which is a rate-limiting enzyme in DNA Current Opinion in Pharmacology 2009, 9:470–481

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synthesis because it converts ribonucleotide diphosphates into deoxyribonucleotide diphosphates. Blocking the ribonucleotide reductase activity, gemcitabine decreases the deoxynucleotide triphosphate pool in the cells, causing a competitively higher incorporation of gemcitabine triphosphate, as compared with dCTP, into the DNA [54]. The mechanisms by which gemcitabine eliminates splenic CD11b+Gr-1+ cells in tumor-bearing mice are not known. One possibility is that gemcitabine induces a massive efflux of MDSCs into restricted anatomical compartments, even though no increase in the number of CD11b+Gr-1+ cells either in the blood or tumors of mice treated with the drugs was ever detected. Another, more attractive possibility is the selective killing of MDSCs. In fact a significantly increased rate of apoptosis in splenocytes at specific time points after in vivo gemcitabine treatment, together with in vitro studies, indicates that gemcitabine accelerates the death of CD11b+Gr-1+ cells without any effects against other cells [52]. Gemcitabine, used either as a single agent or in combination with cisplatin or paclitaxel, has been tested in many clinical trials. In fact, in metastatic breast cancer overexpressing Her-2/neu antigen, an adjuvant therapy based on the combination of gemcitabine, trastuzumab, and docetaxel was shown to be efficacious [55]. Recently, gemcitabine was combined with vaccination with a recombinant adenovirus expressing the tumor associated antigen (TAA) Her-2/neu and the administration of antiglucocorticoid-induced tumor-necrosis factor receptor-related protein (GITR) mAbs, a treatment that was expected to reduce in vivo Treg cell immunosuppressive activity [56]. There is a complex interplay between MDSCs and Treg cells. The ability of MDSCs to promote de novo development of Treg cells in vivo has been documented by few studies. In a mouse model of lymphoma, MDSCs were shown to induce the expansion of antigen-specific, naturally occurring Treg cells through a TGF-b-independent mechanism that involved ARG1 and the capture, processing, and presentation of TAAs by MDSCs [57]. The relationship between MDSCs and Treg cells is not limited to the homeostatic control of CD4+ Treg population. In fact, in a mouse ovarian carcinoma able to accumulate MDSCs and Treg cells in spleen, ascites, and tumor tissue blocking either CD80 or its ligand CD125 significantly retarded tumor growth, suggesting that tumor-mediated CD80 upregulation on MDCSs was a key step for immune evasion and tumor progression. Interestingly, the engagement of CD80 on MDSCs by Treg cell-expressed CD152 was required for MDSCTreg cell cooperation in inhibiting both priming and IFN-g production by antigen-specific T cells. Binding of CD80 and CD152 may thus activate the MDSC suppressive program, suggesting that in some cases MDSCs and not Treg cells might be the final effectors of immune suppression. Current Opinion in Pharmacology 2009, 9:470–481

We recently tested the ability of another chemotherapy drug, 5-Flurouracil (5-FU), to eliminate MDSCs. 5-FU is commonly used for the treatment of several cancers, including colon, breast, stomach, and pancreas cancer. Normally, 5-FU is administered by the intravenous route even though its life span in blood and body tissues is very short and limited to minutes. As a pyrimidine analog, it is transformed inside the cell into different cytotoxic metabolites, which are then incorporated into DNA and RNA, finally inducing cell cycle arrest and apoptosis by inhibiting the cell’s ability to synthesize DNA. We observed that, at low doses, in vivo administration of 5-FU did not arrest tumor progression but caused an important contraction of splenic MDSCs in tumor-bearing mice and restored the immune response against TAAs. In addition, when we combined the 5-FU injection with the adoptive transfer of antigen-specific CD8+ T lymphocytes in tumor-bearing Rag-2/ gc/ mice, we observed a significant synergy in the therapeutic activity on established subcutaneous tumors (S Ugel, manuscript in preparation). These data suggest that MDSCs can be, at least in some tumor models, the main players of tumor-induced immunosuppression, not requiring the help of other cell populations. In fact, Rag-2/ gc/ mice are deficient for all cells of lymphoid-origin, including Treg cells [58]. Another indirect strategy to reduce MDSC accumulation is to interfere with chemokines recruiting them from body deposits, such as the bone marrow and the spleen. This strategy suffers from the redundancy in MDSC chemoattractants produced by different tumors, but some encouraging results have recently been reported. It was shown, in fact, that recruitment of CD11b+Gr-1+ cells to tumor tissues is regulated mainly by two different chemokine axes: SDF-1/CXCR4 and CXCL5/CXCR2. Genetic deletion of type II TGF-b receptor led to the finding that CXCR4 is upregulated in CD11b+Gr-1+ cells, in a tumor type-dependent fashion, and CXCL5 is overexpressed in tumor microenvironment. CD11b+Gr-1+ cells express high levels of CXCR4, which interacts with SDF-1 produced by tumor cells, and thus they are recruited into the tumor microenvironment where they promote local tumor invasion and distant metastasis formation, through the elevated activity of MMP14, MMP13, and MMP2; at the same time, CXCL5 interaction with CXCR2 contributed to a supplementary pathway involved in MDSC recruitment. Interestingly, CXCR2-specific (S-265610) and CXCR4-specific antagonist (AMD3100) induced a drastic reduction of MDSC number in tumor-bearing mice [59]. Although it remains to be determined with how many chemokine/ chemokine receptor axes one needs to interfere in order to have a complete block of MDSC migration to the periphery, it is clear that a pharmacological approach against MDSC chemotaxis might provide additional therapeutic options, such as inhibitions of tumor spread and metastasis. www.sciencedirect.com

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Drugs affecting MDSC inhibitory functions The last approach affecting MDSCs consists in the interference with pathways that directly control the immunoregulatory activity of these cells. As described previously, MDSC immunosuppression is principally achieved by Larginine metabolism through the activity of two enzymes: ARG1 and NOS2 [9]. The effectiveness of removing MDSC inhibitory mechanisms in order to restore T cell responses was proven in several studies, by using either ROS scavengers or selective antagonists for ARG1 and NOS2 (nor-NOHA and l-NMMA, respectively) [12,60,61]. Unfortunately, many of these compounds are basically useless for therapeutic approaches: for example, l-NMMA has an adverse effect when used in large doses, while blocking ARG1 activity with norNOHA would inhibit the urea cycle. Deeper comprehension of MDSC biology is showing us that these cells probably use both ARG1 and NOS2 at the same time to suppress antitumor responses [1,3]. Therefore, drugs which interfere with both enzymes without relevant side effects should be the best candidates for therapeutic treatment of MDSC-induced tumor tolerance. Actually two classes of compounds have shown to possess these requirements: nitroaspirines and phosphodiesterase 5 (PDE5) inhibitors. Nitroaspirin was developed by coupling a NO-releasing moiety to aspirin in order to reduce its gastrointestinal toxicity. This and other related compounds have many biological activities, like reduction of ROS generation, decrease of proinflammatory cytokine production by monocytes, feedback inhibition of NOS2 catalytic activity, and suppression of cancer cell proliferation. In addition, we demonstrated that NO-releasing aspirin can also be very useful as adjuvant for antitumor DNA-based vaccines [62]. Given orally, NO-aspirin strongly potentiates the efficacy of a cancer vaccine based on an endogenous retroviral antigen, which usually induces a weak immune response in the absence of adjuvants and does not result in a significant antitumor activity [62]. The in vivo adjuvant effect of nitroaspirin was demonstrated in both a colon cancer and a mammary carcinoma model. In spite of the pleiotropic biological actions of NO-releasing compounds, the adjuvant effect appeared to depend on the inhibition of MDSC suppressive pathways. While nitroaspirin was very effective in normalizing the immune status of vaccinated tumor-bearing hosts, when used alone in vivo it only led to a slight retardation of tumor growth, and after the end of its administration no statistically different survival rates were observed among treated and untreated groups [62]. These data suggest that nitroaspirin is useful only if combined with active immunotherapy, even though this drug was shown to have an interesting in vitro antiproliferative/proapoptotic activity on several cancer cell lines [63], and in some models of in vivo carcinogenesis [64]. NCX 4016, the NO aspirin isomer we exploited as cancer vaccine adjuvant, www.sciencedirect.com

has not shown adverse effects in clinical trials. We are currently developing new NO-releasing compounds with heightened activities on MDSC suppressive properties. NO-aspirin effect was associated with a profound inhibition of both ARG1 and NOS2 activity in spleen and tumor-associated myeloid cells; while NO released by nitroaspirin was essential for NOS inhibition, the aspirinspacer portion was responsible for the ARG inhibition [62]. The dose at which we observed a meaningful reduction of ARG activity in vitro was too high to hypothesize a direct in vivo activity of the salicylic portion of the compound, so we speculated that nitroaspirin potentially operates indirectly in two ways: either by inhibiting STAT6-mediated signals triggered by Th2 cytokines (like IL-4 and IL-13) required to upregulate ARG1 expression in myeloid cells, or by interfering with an unknown IL-13/IL-4Ra signal pathway necessary for the acquisition of the suppressive phenotype of MDSC [3,9,51,65]. Moreover, nitroaspirin dramatically reduced the nitration of proteins within the tumor environment [62], an indirect but rather significant measure of the reduction in local production of peroxynitrites, which are able to nitrate and inhibit the TCR of tumor-specific lymphocytes [12]. This activity is in line with the antioxidant properties ascribed to this nitroaspirin [66], and could contribute to the overall interference with MDSC-dependent immunosuppression [1,60]. To further strengthen the relevance of the antioxidant action, urea was also shown to revert tumor-induced tolerance by reducing peroxynitrite generation by MDSCs [12]. Another help against MDSC immunosuppression could be provided by PDE5 inhibitors. These drugs are currently in clinical use for the treatment of erectile dysfunction, pulmonary hypertension, and cardiac hypertrophy, and have shown to have proapoptotic activity in human colon carcinoma and chronic lymphocyte leukemia in vitro [67,68]. The administration of a PDE5 inhibitor (sildenafil) delayed tumor outgrowth by 50–70% in immune-competent mice, although none of the animals survived [69]. Experiments in immunedeficient mice together with in vivo cytotoxicity assays clearly demonstrated that the antitumor effect of sildenafil was immune-mediated. This was confirmed by the observation that administration of different PDE5 inhibitors synergized with adoptive cell therapy to delay tumor outgrowth, and that PDE5-inhibitor-treated mice had an increased intratumoral infiltration of CD8+ T cells, compared to untreated mice. Moreover, the infiltrating lymphocytes upregulated expression of activation markers CD69 and CD25 and secreted IL-2 [69]. The improvement of antitumor immunity was due to the downregulation of MDSC suppressive pathways by PDE5 inhibitors, because sildenafil-treated mice MDSCs showed a decrease in IL-4-Ra expression and downregulation of both ARG1 and NOS2 [69]. Interestingly for Current Opinion in Pharmacology 2009, 9:470–481

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Figure 1

Therapeutic strategies to target MDSCs. MDSC inhibitors can be divided into four classes according to their ability to control: MDSC maturation from precursor cells (blue area); MDSC accumulation in peripheral organs (violet area); MDSC function (yellow area); and MDSC differentiation in mature cells (green area). Specific drugs are indicated by numbers and main properties reported in the enclosed table.

future translation to cancer-bearing patients, PDE5 inhibitors restored T cell proliferation in PBMC isolated from head and neck cancer or multiple myeloma patients [69]. This last experiment, together with other evidences [61], suggests that ARG1 and NOS2 are important in immunosuppression by human MDSCs as well, at least in Current Opinion in Pharmacology 2009, 9:470–481

some types of tumors, and confirms the rationale of testing PDE5 inhibitors as adjuvants in active and passive tumor immunotherapy. PDE5 inhibition causes an increase in intracellular levels of cGMP, reducing the levels of the ubiquitous mRNA www.sciencedirect.com

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binding protein human antigen R, which stabilizes NOS2 mRNA and is reduced by cGMP [70]. This could explain the effect of PDE5 inhibitors on NOS2 expression. By contrast, ARG1 mRNA does not seem to be stabilized by human antigen R, so other mechanisms are likely involved in its downregulation by PDE5 inhibitors. One possible explanation is that the increase in cGMP levels reduces the cytosolic Ca2+ concentration [71], determining a reduction of the calcium-dependent protein kinase C activity, which is required for IL-4Ra expression [72]. IL-4Ra signaling is critical for upregulation of ARG1, as previously described. The importance of the IL-13/IL-4Ra/STAT6 pathway for MDSC suppressive functions opens the possibility for other therapeutic strategies. One of these could be the use of drugs which interfere with IL-4/IL-13 signaling. Such molecules have already been developed for the treatment of asthma and allergies, and consist of modified forms of IL-4 and IL-13 (without biological activity or coupled to toxins) or soluble forms of their receptors, which either impede the signaling mediated by normal IL-4 and IL-13 or kill cells expressing their receptors [73]. These molecules are currently in clinical trials for the treatment of various pathologies, and could be further tested to improve antitumor immune responses. Finally, another target for subverting MDSC-dependent immunosuppression might be cyclooxigenase 2 (COX-2). This enzyme is necessary for the production of prostaglandin E2, which in turn induces ARG1 expression by MDSCs in some experimental mouse tumors [1]. A selective COX-2 inhibitor (Colecoxib) partially prevented the 1,2-dimethylhydrazine-mediated induction of large intestinal tumors in a model of chemical carcinogenesis [74]. This result was linked to the ability of Colecoxib administration to reduce NOS2 and ARG expression together with CD11b+Gr-1+ cell numbers in the spleen of treated mice, as compared with untreated autochthonous tumor-bearing mice, and restore both frequency and function of splenic CD4+ T cells [74].

strated alone. Therefore it is clear that the synergy between an effective immunotherapy approach and an efficient MDSC inhibitor is, at present, the most promising antitumor strategy and it may be translated to other diseases in which the therapeutic aim is the control of MDSC-induced immune dysfunctions. Moreover, the development of new treatments will also help to better clarify the role of MDSCs in tumors of different derivation as well as its importance in various stages of cancer progression.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 

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Conclusions Among the immunosuppressive cell populations induced by cancer MDSCs play a dominant role. MDSCs, in fact, establish tolerance and immune suppression in both tumor microenvironment and in secondary lymphoid organs, promote angiogenesis, and sustain overall tumor growth, proliferation, and spreading. Controlling MDSC recruitment as well as interfering with the molecular pathways of MDSC differentiation/expansion, or blocking MDSC suppressive functions represents a multifaceted approach for the therapy of cancer. Several drugs, summarized in Figure 1, have shown the capability to inactivate MDSCs, but the therapeutic effects of many of these compounds are clear only when they are used in combination with immunotherapy and not when adminiwww.sciencedirect.com

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Current Opinion in Pharmacology 2009, 9:470–481