Direct Effects of Type I Interferons on Cells of the Immune System

Published OnlineFirst March 3, 2011; DOI: 10.1158/1078-0432.CCR-10-1114

Clinical Cancer Molecular Pathways Research

Sandra Hervas-Stubbs1, Jose Luis Perez-Gracia2, Ana Rouzaut3, Miguel F. Sanmamed1,2, Agnes Le Bon4,5, and Ignacio Melero1,2

Abstract Type I interferons (IFN-I) are well-known inducers of tumor cell apoptosis and antiangiogenesis via signaling through a common receptor interferon alpha receptor (IFNAR). IFNAR induces the Janus activated kinase–signal transducer and activation of transcription (JAK-STAT) pathway in most cells, along with other biochemical pathways that may differentially operate, depending on the responding cell subset, and jointly control a large collection of genes. IFNs-I were found to systemically activate natural killer (NK) cell activity. Recently, mouse experiments have shown that IFNs-I directly activate other cells of the immune system, such as antigen-presenting dendritic cells (DC) and CD4 and CD8 T cells. Signaling through the IFNAR in T cells is critical for the acquisition of effector functions. Cross-talk between IFNAR and the pathways turned on by other surface lymphocyte receptors has been described. Importantly, IFNs-I also increase antigen presentation of the tumor cells to be recognized by T lymphocytes. These IFN-driven immunostimulatory pathways offer opportunities to devise combinatorial immunotherapy strategies. Clin Cancer Res; 17(9); 2619–27. 2011 AACR.

Background nals through a receptor complex consisting of interleukin- 10 receptor 2 subunit (IL-10R2) and IL-28 receptor alpha Types of interferon subunit (IL-28Ra; ref. 3). It is well established that IFNs Interferons (IFN) were discovered in 1957 by Isaacs and induce the expression of hundreds of genes, which mediate Lindenmann as soluble proteins able to inhibit virus repli- various biological responses. Some of these genes are regu- cation in cell cultures (1). There are 3 types of IFNs: type I lated by the 3 classes of IFNs, whereas others are selectively (IFN-I), type II (IFN-II), and type III (IFN-III; refs. 2, 3). IFNs regulated by distinct IFNs. belonging to all IFN classes are very important for fighting Although the role of IFN-II and IFN-III is very important viral infection. In humans, IFNs-I include IFN-a subtypes, in immune responses, IFN-II has not yet shown any clinical IFN-b, IFN-e, IFN-k, and IFN-w (2). From an immunologic activity for human cancer treatment (4), and IFN-III is not perspective, IFN-a and IFN-b are the main IFN-I subtypes of currently developed for any indication (3). However, since interest. Both are produced by almost every cell of the body the discovery of IFNs, the use of IFN-I in therapy has been in response to viral infection and share important antiviral very widespread. In fact, systemic injections of IFN-I are properties. The IFN-a family is a multigenic group of highly approved for the treatment of a variety of diseases, which homologous polypeptides encoded by more than 13 include solid and hematologic malignancies, multiple intronless genes in humans. IFN-b, in contrast, exists as a sclerosis, and, above all, chronic viral hepatitis. In this single gene in most species. IFN-g is the only IFN-II. This review, we focus on the role of IFN-I as a direct immunos- cytokine does not have marked structural homology with timulatory agent directly modifying functions of immune IFNs-I and binds a different cell-surface receptor (IFNGR), system cells and how these functions can be applied to also composed of 2 different subunits (IFNGR1 and better therapeutic strategies. IFNGR2; ref. 2). The recently classified IFN-III consists of 3 IFN-l molecules (IFN-l1, IFN-l2, and IFN-l3) and sig- IFN-I signaling pathways All types of IFN-I signal through a unique heterodimeric receptor, interferon alpha receptor (IFNAR), composed of 2 1 Authors' Affiliation: Division of Gene Therapy and Hepatology, Centre for subunits, IFNAR1 and IFNAR2, which are expressed in Applied Medical Research (CIMA), University of Navarra, Pamplona, 2Medical Oncology Department and Cell Therapy Unit, University Clinic, most tissues (5). IFNAR1 knockout mice have not only University of Navarra, and 3Division of Oncology, CIMA, University of confirmed the role this subunit plays in mediating the 4 Navarra, Pamplona, Spain; Institut Cochin, Universite Paris Descartes, biological response to IFNs-I, but have also established CNRS (UMR 8104), and 5INSERM, U1016, Paris, France the pleiotropic role that these IFNs play in regulating the Corresponding Author: Ignacio Melero, CIMA and Facultad de Medicina Universidad de Navarra, Av. Pio XII, 57, 31008 Pamplona, Spain. Phone: host response to viral infections and in adaptive immunity 34-948194700; Fax: 34-948194717; E-mail: [email protected] or msher- (6–8). [email protected] Receptor occupancy rapidly triggers several signaling doi: 10.1158/1078-0432.CCR-10-1114 cascades (Fig. 1), which culminate in the transcriptional 2011 American Association for Cancer Research. regulation of hundreds of IFN-stimulated genes (ISG). The

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Type I IFNs signaling pathways (A) JAK-STAT (B) CRK (C) PI3K and NF␬B (D) MAPK ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ CD45 IFN-␣/␤ IFN- / IFN- / IFN- / IFN- / TCR complex ␣␤ CD4/8 ␦␧␧␥ IFNAR1 IFNAR2 IFNAR1 IFNAR2 IFNAR1 IFNAR2 IFNAR1 IFNAR2 IFNAR1 IFNAR2 ␨␨

Tyk2 JAK1 Tyk2 JAK1 Tyk2 JAK1 Tyk2 JAK1 STAT STAT VAV STAT STAT STAT CRKL TRAF Lck 5 3 3 SLP76 Zap70

NIK LAT C3G RS1/2 VAV PI3K mTOR Inhibition of Homodimers AKT GSK-3/CDKN1A/ STAT STAT STAT1, STAT3 CDKN1B VAV STAT4, STAT5 C3G STAT CRKL STAT6 5 Heterodimers IKK␣ IKK␤ PKC␪ Ras Rac1 STAT STAT STAT STAT Rap1 Raf1 1 2 STAT1/2,3,4 or 5 STAT2/3 Proliferation NF␬B MEK1/2 STAT5/6 Differentiation p38 JNK IRF9 Survival .... ERK1/2

Homodimers Fos MEF2 Jun NFAT c-Myc STAT STAT Elk-1 MAPKAP Elk-1 Ets SAP-1 Sap-1 Rsk NFAT Elk-1 P53 IRF9 Heterodimers ATF2 MEF2 SP1 Stat1/3 SMADs STAT STAT STAT CRKL 1 2 STAT STAT 5 NF␬B Rel ...... ISRE GAS GAS ISG15 ISG15, IP-10, IRF-1, IRF-2, Regulated Regulation of expression of Histone acetylation Apoptosis Cellular growth IRF-7, PKR, IRF-8, IRF-9, genes or • GTP-binding protein Antiviral effect Differentiation 2’-5’-OAS, etc functions • Ag processing/presentation protein Growth inhibitory effects Ser phosphorylation etc Survival signals Hematopoietic suppressive signals of STAT1

Figure 1. Signaling pathways activated by the IFN-I receptor. Schematic of signaling pathways downstream of the IFNAR, emphasizing cross-talk with different signaling cascades, which depends on the identity and activation status of the cell responding to IFN-I. A, JAK-STAT signal pathway activated by IFNs-I. B, CRKL pathway. Upon JAK activation, CRKL associates with TYK2 and undergoes tyrosine phosphorylation. The activated form of CRKL forms a complex with STAT5, which also undergoes TYK2-dependent tyrosine phosphorylation. The CRKL–STAT5 complex translocates to the nucleus and binds specific GAS elements. C, PI3K and NF-kB pathways. Phosphorylation of TYK2 and JAK1 results in activation of PI3K and AKT, which in turn mediates downstream activation of mTOR, inactivation of GSK-3, CDKN1A, and CDKN1B, and activation of IKKb, which results in activation of NF-kB. IFNs-I also activate NF-kB by an alternative pathway that involves the linkage of TRAFs, which results in activation of NF-kB2. PI3K/AKT can also activate NF-kB through an activation loop via PKCq. D, MAPK pathway. Activation of JAK results in tyrosine phosphorylation of Vav, which leads to the activation of several MAPKs such as p38, JNK, and ERK1/2. The different MAPKs activate different sets of transcription factors, such as Fos, ELK1, Sap-1, MEF2, MAPKAP, and Rsk (activated by p38); Jun, ELK1, NFAT, and ATF2 (activated by JNK); and NFAT, ETS, ELK1, MEF2, STAT1/2, c-Myc, SAP-1, p53, SP1, and SMADs (activated by ERK1/2).

first signaling pathway shown to be activated by IFNs-I was STAT2 are both phosphorylated on a single tyrosine the Janus activated kinase–signal transducer and activation (Y701 for STAT1 and Y690 for STAT2). Once phosphory- of transcription (JAK-STAT) pathway (Fig. 1A; ref. 9), which lated, STATs dimerize and move to the nucleus, where they is active in almost all cell types. IFNAR1 is constitutively associate with the IFN-regulatory factor 9 (IRF9) to form associated with tyrosine kinase 2 (TYK2), whereas IFNAR2 the ISG factor 3 (ISGF3) transcription factor complex, is associated with JAK1. Receptor binding results in trans- which binds to IFN-stimulated response elements. phosphorylation of JAK1 and TYK2. The activated JAKs Although STAT1 and STAT2 are the most important then phosphorylate conserved tyrosine residues in the mediators of the response to IFNs-I, other STATs, namely cytoplasmic tails of the IFNAR. These phosphotyrosyl resi- STAT3 and STAT5, have been found to be phosphorylated dues subsequently serve as docking sites for the recruitment (activated) by IFNs-I (Fig. 1A; refs. 10–13). STAT4 of src-homology 2 (SH2)–containing signaling molecules, and STAT6 can also be activated by IFN-a; however, this such as STAT1 and STAT2. Once at the receptor, the STAT activation seems to be restricted to certain cell types, such proteins themselves become JAK substrates. STAT1 and as endothelial or lymphoid cells (14–17). Following

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phosphorylation, activated STATs form homo- (STAT1, such as glycogen synthase kinase 3 (GSK-3) and cyclin STAT3, STAT4, STAT5, and STAT6) or heterodimers dependent kinase inhibitors 1A and 1B (CDKN1A and (STAT1/2, STAT1/3, STAT1/4, STAT1/5, STAT2/3, and CDKN1B), all of them involved in the control of cell STAT5/6; refs. 10, 11, 18, 19), which translocate to the division and proliferation. The AKT pathway also results nucleus and bind other regulatory sequences known as in the activation of IkBkinasebeta(IKKb), which gives IFN-g–activated sites (GAS). rise to NF-kB activation. IFNs-I also activate NF-kBbyan Interestingly, distinct STATs have opposing biological alternative pathway, which involves the linkage of TNF effects. Thus, STAT3 stimulates growth, whereas on the receptor–associated factors (TRAF) to the activation of contrary, STAT1 is a growth inhibitor. On the other hand, the NF-kB–inducing kinase (NIK), which in turn results IFNs-I–mediated activation of STAT4 is required for IFN-g in activation of NF-kB2. This alternative pathway is production, whereas surprisingly, STAT1 negatively regu- strictly dependent on the activity of IKKa. In addition, lates the IFNs-I–dependent induction of IFN-g (20). The PI3K/AKT can also activate NF-kB through an activation relative abundance of these dimeric transcription factors, loopviaproteinkinaseCzeta(PKCq). Overall, NF-kB which may vary substantially depending on cell type and activated by IFNs-I regulates prosurvival signals and activation and/or differentiation state, is likely to have a enhances the expression of several GTP-binding proteins major impact on the overall response to IFNs-I (21–23). as well as molecules involved in antigen processing and/ Stimulation of the IFNAR/JAK/STAT1 pathway can also or presentation. lead to the upregulation of the TAM receptor Axl, which The MAPK pathway. Activation of JAK results in tyro- accumulates at the cell surface where it physically associates sine phosphorylation of the guanine nucleotide exchange with the IFNAR and usurps the IFNAR-STAT1 pathway by factor Vav, which leads to downstream activation of Rat stimulating the transcription of suppressors of cytokine sarcoma protein (Ras) and ras-related C3 botulinum toxin signaling (SOCS; refs. 24, 25). The SOCS proteins extin- substrate 1 (Rac1), which subsequently leads to the activa- guish IFN-I signaling in a negative feedback loop. For tion of several MAPKs: p38, c-Jun N-terminal kinases instance, SOCS1 directly interacts with JAKs blocking their (JNK), and extracellular signal regulated kinases (ERK) 1 catalytic activity, whereas SOCS3 seems to inhibit JAKs after and 2 (9). Vav can also activate PKCq, leading to the gaining access by receptor binding. In addition, SOCS activation of the NF-kB cascade. Several studies have shown proteins interact with the cellular ubiquitination machin- that IFN-a–mediated activation of ERK1/2 in T lympho- ery through the SOCS-box region and may redirect asso- cytes requires proteins involved in the T-cell receptor (TCR) ciated proteins, such as JAKs or IFNAR chains, to early signaling complex, such as CD45, lymphocyte-speci- proteasomal degradation. It has been shown that SOCS1 fic protein tyrosine kinase (Lck), Zeta-chain-associated knockout mice develop lupus-like disease symptoms (26) protein kinase 70 (Zap70), SH2 domain-containing leu- and that SOCS1 silencing enhances the antitumor activity kocyte protein of 76 kDa (SLP76), and Vav1, and possibly of IFNs-I (27), suggesting that SOCS are key elements in linker for activation of T cells (LAT), indicating cross-talk keeping IFNs-I effects under control. between pathways (29–31). Moreover, it has been shown recently that TCR deletion in Jurkat cells abrogated IFNAR- Alternative signaling pathways and cross-talk with stimulated MAPK activity, whereas the canonical JAK-STAT other receptors pathway remained unaffected (31). A hypothetical path- Evidence is accumulating that several other signaling way that involves all these molecules and results in Vav elements and cascades are required for the generation of activation is depicted in Fig. 1D. The different MAPKs many of the responses to IFNs-I, such as the v-crk sarcoma activate different sets of transcription factors. Importantly, virus CT10 oncogene homolog (avian)-like (CRKL) path- p38 functions are essential for the antiviral and antileuke- way, the mitogen activated protein kinase (MAPK) path- mic properties of IFNs-I, it plays a role in the hematopoie- way, the phosphoinositide 3-kinase (PI3K) pathway, and tic-suppressive signals and is required for the growth- either the classical or the alternative NF-kB cascade inhibitory effects of IFNs-I observed in certain lymphocyte (Fig. 1B-D; refs. 9, 28). Some of these pathways operate cultures. IFNs-I–activated ERK cascades regulate cellular independently of the JAK-STAT, whereas others cooperate growth, differentiation, and serine phosphorylation of with STATs to tune the response to IFNs-I. Interestingly, STAT1. The search for biochemical signals to explain the MAPK, PI3K, and NF-kB pathways involve immunorecep- effects of IFN-I on immune system cells is far from over. tor tyrosine-based activation motives used by classical immunoreceptors, suggesting a cross-talk among IFNAR Effects of IFNs-I on cells of the immune system and multiple receptors on immune system cells. It has long been established that the main mechanism The PI3K and NF-kB pathways. Upon activation, TYK2 accounting for the efficacy of the IFN-I–based therapies was and JAK1 phosphorylate insulin receptor substrate 1 (IRS1) due to their direct effect on malignant or virus-infected and 2 (IRS2), which provide docking sites for the PI3K (9). cells. In fact, IFNs-I directly inhibit the proliferation of STAT3 acts as an adapter to couple PI3K to the IFNAR1 tumor and virus-infected cells and increase MHC class I subunit. PI3K subsequently activates the serine-threonine expression, enhancing antigen recognition. Moreover, kinase AKT, which in turn mediates downstream the acti- IFNs-I repress oncogene expression and induce that of vation of mTOR and the inactivation of several proteins, tumor suppressor genes, which may contribute to the

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inhibitory effects of IFN-I on malignant cells in conjunc- concentrations of IFNs-I are essential for the optimal pro- tion with the antiangiogenic effects. duction of IL-12p70 (46), whereas higher levels of IFN-I IFNs-I have also proven to be involved in immune suppress IL-12p40 expression, thus dampening IL-12p70 system regulation (7, 8, 32). IFNs-I exert their effects on production (47). immune cells either directly, through IFNAR triggering, or A key requisite to initiate the adaptive immune response indirectly (i) by the induction of chemokines, which allow is the arrival of professional APC from infected tissue into the recruitment of immune cells to the site of infection; (ii) lymph nodes. It has been shown that human DCs differ- by the secretion of a second wave of cytokines, which could entiated from monocytes in the presence of IFN-a exhibit further regulate cell numbers and activities (as for example upregulation of CC chemokine receptor type 7 (CCR7), IL-15, which plays a critical role in proliferation and correlating with an enhanced chemotactic response in vitro maintenance of NK cells and memory CD8 T cells; refs. to CCR7 natural ligand (CCL19) and with strong migratory 33, 34); or (iii) by the stimulation of other cell types critical behavior in SCID mice (48). Interestingly, experiments in for the activation of certain immune cells, such as DC for mice have also shown that IFN-a/b is required for plasma- the activation of na€ve T cells. cytoid DCs (pDC) to migrate from the marginal zone into One of the earliest described immunoregulatory func- the T-cell area, where they form clusters (49). We have also tions of IFNs-I was their ability to regulate NK functions recently shown that IFNs-I enhance the adhesion and (32). IFNs-I enhance the ability of NK cells to kill target extravasations of DCs across inflamed lymphatic cells and to produce IFN-g by indirect and direct mechan- endothelium in a lymphocyte function-associated anti- isms (33, 35, 36). Furthermore, IFNs-I promote the accu- gen 1 (LFA-1)– and very late antigen-4 (VLA4)–depen- mulation and/or survival of proliferating NK cells by the dent fashion (50). IFNs-I may also favor the encounter IFN-I/STAT1–dependent induction of IL-15 (33). IFNs-I between DCs and specific lymphocytes in lymph nodes also enhance the production or secretion of other cytokines by promoting lymphocyte trapping in lymph nodes upon by the NK cell through the autocrine IFN-g loop (37). downmodulation of sphingosine 1-phosphate (51). IFNs-I also affect monocyte and/or macrophage function IFNs-I have also been shown to potently enhance the and differentiation. Thus, IFNs-I markedly support the primary antibody response to soluble antigen, stimulating differentiation of monocytes into DC with high capacity the production of all subtypes of immunoglobulin G (IgG), for Ag presentation, stimulate macrophage antibody- and inducing long-lived antibody production and immu- dependent cytotoxicity, and positively or negatively regu- nologic memory (8). Direct effects of IFN-a on DCs (8), B late the production of various cytokines (e.g., TNF, IL-1, IL- cells (52, 53), and CD4 T cells (53) all contribute to its 6, IL-8, IL-12, and IL-18) by macrophages (38). In addition, adjuvant activities on humoral immune responses. Inter- autocrine IFN-I is required for the enhancement of macro- estingly, a direct effect of IFNs-I on B cells seems to be phage phagocytosis by macrophage colony-stimulating crucial for the development of local humoral responses factor and IL-4 (39) and for the lipopolysaccharide-, against viruses (54). virus-, and IFN-g–induced oxidative burst through the As mentioned above, CD4 T cells are direct targets of IFN- generation of nitric oxide synthase 2. I in the enhancement of antibody responses (53). In The main function of DCs is to uptake and process humans, it has also been described that direct effects of antigens for presentation to T cells. DCs are a unique cell IFN-I on naive CD4 T cells favor their differentiation into type able to prime na€ve T cells and, therefore, are critical IFN-g–secreting Th1-like T cells (55). antigen presenting cells (APC). IFNs-I have multiple effects Several studies suggested that IFNs-I might exert a direct on DCs, affecting their differentiation, maturation, and effect on CD8 T cells. Thus, it was reported that IFNs-I migration. Thus, human monocytes cultured in granulo- promote IFN-g production by CD8 T cells in a STAT4- cyte-macrophage colony-stimulating factor (GM-CSF) þ dependent manner (56) and promote survival of CD8 T IFN-a/b, rather than the more commonly used combina- cells from wild-type (WT) but not IFNAR-deficient tion of GM-CSF þ IL-4, differentiate more rapidly into DCs, (IFNAR / ) mice (57). The definitive report showing that exhibiting the phenotype of partially mature DCs, while CD8 T cells represent direct targets of IFN-I–mediated showing a strong capability to induce a primary human stimulation in vivo came from Kolumam and colleagues antibody response and CTL expansion when pulsed with (58). By adoptively transferring virus-specific naive CD8 T antigen and injected into humanized severe combined cells from IFNAR / or IFNAR-sufficient mice into normal immunodeficiency (SCID) mice (40–42). IFNAR WT hosts, IFNs-I were shown to act directly on In vitro treatment of immature conventional DCs with murine CD8 T cells, allowing their clonal expansion and IFN-a/b has been shown to upregulate surface expression memory differentiation. Subsequent studies confirmed this of MHC class I, class II, CD40, CD80, CD86, and CD83 work (59–61). Elegant experiments in mice by Curtsinger molecules in the human system (43–45), associated with a and colleagues (62) have shown that, in addition to signals heightened capacity to induce CD8 T-cell responses. via TCR (signal-1) and CD28 (signal-2), na€ve CD8 T cells Accordingly, it has been observed in mice that IFN-a acting required a third signal to differentiate into effector cells. on DC plays a key role in achieving efficient cross-priming cDNA microarray analyses showed that IFN-a could of antigen-specific CD8 T lymphocytes (7, 8, 32). IFNs-I regulate critical genes involved in CTL functions (63), also affect the ability of DC to secrete IL-12p70. Low providing evidence that IFNa promoted activation and

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differentiation of CD8 T cells by sustaining the expression efficacious when acting locally and intermittently at the of T-bet and Eomes through chromatin remodeling. malignant tissue and tumor-draining lymph node than Recently, we have shown that IFN-a provides a strong when administered to achieve systemic bioavailability. and direct signal to human CD8 T cells, thereby resulting Local delivery can be achieved with pharmaceutical for- in upregulation of critical genes for cytotoxic T-cell activity mulations and gene therapy approaches (70). In our (cytolysis and IFN-g secretion) and for the production of opinion, the clinical use of recombinant IFN-a as a vaccine chemokines that would coattract other effector lympho- adjuvant should be reconsidered and new investigations cytes. IFN-a was absolutely critical in the case of human carried out with an eye to clinical applications (71). na€ve CD8 T cells for effector function acquisition (64). In The rationale for using intermittent delivery arises from many instances, T cells may sense IFN-I before the cognate observations indicating that IFN-I turns on signaling desen- antigen is actually presented to them. As a result of a sitizing mechanisms (i.e., because of SOCS1 induction). preexposure to IFN-I, CD8 T cells become preactivated Intermittency at an optimized pace may help to avoid these and acquire much faster effector functions upon antigen negative feedback mechanisms in the responding immune recognition (65). This preactivation may reflect the upre- cells. These ideas contrast with stabilized formulations, gulation by IFN-I of several mRNAs in antigen-naive CD8 T such as the widely used polyethylene glycol-conjugated lymphocyte, albeit without changes in the expression of the IFN-a and fusion proteins with albumin that extend the corresponding protein, unless antigen-specific activation half-life of the cytokine. ensues (64). It is of much interest that a gene expression signature induced by IFN-I in melanoma lesions predicts benefit Clinical-Translational Advances from vaccines and may have a key role in recruiting effector T cells to tumors by inducing chemokine genes The contracting current indications of IFN-a as an (72). IFN-a has been recently used in promising combi- anticancer agent natorial immunotherapy approaches alongside DC vac- IFN-a has received approval for treatment of several cination for glioblastoma, precisely to promote T-cell neoplastic diseases (66). The most frequent indications infiltration and DC activation(73).Inthisregard,using for IFN-a are chronic viral hepatitis. For the treatment of endogenous IFN-I inducers instead of recombinant cyto- these infectious conditions, stabilized forms of IFN-a have kines may have advantages, because substances such as been created by directed conjugation of polyethylene gly- the viral RNA analog poly I:C will also elicit other cyto- col, which, by increasing molecular weight, retards renal kines that may act in concert with IFNs-I to enhance the clearance and, thereby, extends plasma concentrations with antitumor immune response. sustained receptor occupancy (67). However, PEG-conju- Creative combination strategies are building on the gated IFN is not widely used in oncology in the absence of knowledge that IFN-a effects on the immune system comparative clinical studies with the unconjugated form. are likely to improve its therapeutic profile against malig- In oncology, the main indication of IFN-a is for patients nant diseases. For instance, combinations with immu- with resected stage II and III melanoma, in whom IFN-a nostimulatory monoclonal antibodies, IL-15, IL-12, and prolongs disease-free survival and shows a trend toward IL-21, may hold much promise. Indeed, intratumoral increased overall survival (68). However, the advent of releaseofmouseIFN-a along with systemic treatment CTLA-4 antagonist antibodies will very likely displace with anti-CD137 mAb results in synergistic therapeutic the use of IFN-a for melanoma therapy (69). effects (74). Macrophages homing to tumor, transduced Table 1 shows the spectrum of neoplastic diseases in which to express IFN-a, exert therapeutic effects without the IFN-a has been used. As shown, its role in cancer therapy is systemic side effects (75). These studies suggest that local steadily decreasing because of its limited efficacy and the delivery as opposed to systemic administration should be advent of new, more effective, and/or safer treatments. tested. Of great importance is the notion that the chemokines induced by IFN-I (i.e., CXCL9, CXCL10) attract Future perspectives activated lymphocytes, thereby calling more immune IFNs-I are powerful tools to directly and indirectly cells to infiltrate the malignant focus. modulate the functions of the immune system. Evolution When reflecting on the use of IFN-I for cancer, we must has shaped these cytokines as a key sign of alarm in case think about reprogramming the tumor microenviron- of viral infection. The type of immune response that we ment, frequently devoid of any stimulatory signals. Local would like to induce and sustain against cancer antigens IFN-I release with concomitant irradiation and chemo- is identical to the one we commonly observe upon acute therapy may help to turn some of the macroscopic tumor viral infections. Obviously, the effects of IFN-I are inte- lesions into immunogenic vaccines. Factors such as CpG grated with many other key molecules that need to act in a oligonucleotides or poly I:C that induce the release of concerted fashion. endogenous IFNs-I at a given tumor lesion (76) are As described in Table 1, side effects of systemic long- promising in this regard. These agents may constitute term treatments and lack of sufficiently high efficacy have an advantageous alternative because these agents also dampened the interest of IFN-a for clinical use in oncol- induce an additional array of proinflammatory mediators ogy. However, we believe that IFN-a is likely to be more acting in synergy with IFNs-I.

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Table 1. Contracting Indications of IFN-a for Malignant Conditions

Indication Stage Comment Standard Current Therapy Reference

Melanoma II/III Prolongs disease-free IFN-a is the standard option. 68 survival and shows a trend toward increased overall survival. IV Combination with Chemotherapy. The advent 77, 78 IL2: Increased response of CTLA-4 antagonist antibody rates but no survival will displace the use of IFN-a improvement. for melanoma inmunotherapy. RCC II/III Two phase III trials showed Novel targeted agents such 79, 80 negative results. as sunitinib, temsirolimus, sorafenib, or everolimus have decreased the use of IFN-a to a minimum in both resectable and advanced RCC. IV Widely studied as a single 81 agent or in combination with IL-2, chemotherapy, or both: modest or negative results. Hairy cell leukemia First treatment that showed Purine analogs are now 82, 83 clinical benefit in this disease. considered standard treatment for this disease. Recommended, in low dose, as a therapeutic option for maintenance therapy and as an alternative for frail patients. Multiple myeloma Extensively studied as Largely replaced by newer 84 maintenance treatment, and more effective agents as single treatment, or in such as thalidomide, combination with lenalidomide, or bortezomib. chemotherapy: slight efficacy. CMS PV Optional treatment for The mainstay of therapy 85–87 cytoreduction (94.6% remains repeated phlebotomy. complete responses). ET Treatment of choice Hydroxyurea. Anagrelide. 86 in women with childbearing potential and optional treatment in young patients resistant to hydroxyurea. PM Cytoreduction therapy. Hydroxyurea. Thalidomide. 77 CML First-line therapy in Imatinib, in a phase III trial, 88–92 combination with has been superior in cytarabine: 71% clinical tolerability, complete response rate and 39% major hematologic and cytogenetic cytogenetic response rate. response rates, and progression-free survival.

(Continued on the following page)

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Table 1. Contracting Indications of IFN- for Malignant Conditions (cont'd )

Indication Stage Comment Standard Current Therapy Reference

In comparison with In imatinib resistance, IFNa chemotherapy IFNa has been precluded by the has been shown to appearance be superior. of dasatinib. Hemangioma Complicate hemangiomas that Steroids. 93 do not respond to steroids. Rate regression in 58% of the patients. AIDS-related Kaposi Evidence for clinical activity. Liposomal anthracyclines are 94 sarcoma preferred as the first treatment option.

PV, polycythemia vera; ET, essential thrombocythemia; PM, primary myelofibrosis; RCC, renal cell cancer; CMS, chronic myelo- proliferative syndrome; CML, chronic myeloid leukemia

In conclusion, the notion of key direct effects of IFNs-I Acknowledgments on immune system cells and detailed knowledge on the elicited signaling pathways should help biomarker discov- We are grateful for continuous scientific collaboration on IFNs-I with Jesus Prieto, Esther Larrea, Jose I. Riezu-Boj, Iranzu Gonzalez, and Juan Ruiz. ery to identify the small number of patients who could actually benefit from current treatments. More importantly, these ideas might change the way we use IFN-a for cancer Grant Support therapy, suggesting new strategies and combinations with MEC/MCI (SAF2005–03131, SAF2008–03294, and TRA2009–0030), Departa- other agents. mento de Educacion y Departamento de Salud (Beca Ortiz de Landazuri) del Gobierno de Navarra. Redes tematicas de investigacion cooperativa RETIC (RD06/0020/0065), Fondo de investigacion sanitaria (FIS PI060932), European commission VII framework program (ENCITE), Immunonet SUDOE. Fundacion Disclosure of Potential Conflicts of Interest Mutua Madrileña, and "UTE for project FIMA". S. Hervas-Stubbs was supported by AECC and by MCI (RYC-2007–00928). I. Melero, A. Rouzaut, and S. Hervas-Stubbs receive laboratory reagents and research funding from Digna Biotech. I. Melero has acted as a consultant Received October 22, 2010; revised January 17, 2011; accepted January for Bristol-Myers Squibb and Pfizer Inc. 17, 2011; published OnlineFirst March 3, 2011.

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Direct Effects of Type I Interferons on Cells of the Immune System

Sandra Hervas-Stubbs, Jose Luis Perez-Gracia, Ana Rouzaut, et al.

Clin Cancer Res 2011;17:2619-2627. Published OnlineFirst March 3, 2011.

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