Molecular Pathways - Clinical Cancer...

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Molecular Pathways

Direct Effects of type I IFNs on cells of the immune system

Sandra Hervas-Stubbs1, Jose Luis Perez-Gracia2, Ana Rouzaut3, Miguel Fernandez de

Sanmamed1,2, Agnes Le Bon4,5 and Ignacio Melero1,2

1Division of Gene Therapy and Hepatology, Centre for Applied Medical Research

(CIMA), University of Navarra, Pamplona 31008, Spain

2Medical Oncology Department and cell therapy Unit, University Clinic, University of

Navarra, Pamplona 31008, Spain

3Division of Oncology, CIMA, University of Navarra, Pamplona 31008, Spain

4Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France

5INSERM, U1016, Paris, France

Correpondence :

Ignacio Melero or Sandra Hervas-Stubbs

Av. Pio XII, 57

31008 Pamplona

Spain

[email protected] or [email protected]

Abbreviations:

Type I interferons: IFNs-I; Interferon alpha Receptor: IFNAR; JAK-STAT: Janus

Activated Kinase-Signal Transducer and Activation of Transcription; Natural killer:

Research. on September 20, 2018. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 3, 2011; DOI: 10.1158/1078-0432.CCR-10-1114

http://clincancerres.aacrjournals.org/

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NK; Dendritic cell: DC; Interferon-γ receptor: IFNGR; Interleukin: IL IFN-stimulated

genes: ISGs; Tyrosine kinase 2: TYK2; Src-homology 2: SH2; IFN-regulatory factor 9:

IRF9; ISG factor 3: ISGF3; IFN-stimulated response elements : ISREs; IFNγ-activated

sites: GAS; SOCS: suppressors of cytokine signaling; v-crk sarcoma virus CT10

oncogene homolog (avian)-like: CRKL; Mitogen-activated protein Kinase: MAPK;

Phosphatidylinositol 3-kinase: PI3K; Nuclear Factor kappa B: NF-κB; Insulin receptor

substrate: IRS1; Mammalian target of rapamycin: MTOR; Glycogen synthase kinase 3:

GSK-3; Cyclin dependent kinase inhibitors 1A and 1B: CDKN1A and CDKN1B; IκB

kinase: IKK; Tumor necrosis factor receptor associated factors: TRAFs; NF-kB-

inducing kinase: NIK; Protein kinase C zeta: PKCθ; Guanosine triphosphate: GTP; Rat

Sarcoma protein: Ras; Ras-related C3 botulinum toxin substrate: Rac; c-Jun N-terminal

kinases: JNK; Extracellular signal-regulated kinases: ERK; T cell receptor: TCR;

Lymphocyte-specific protein tyrosine kinase: Lck; Zeta-chain-associated protein kinase

70: Zap70; SH2 domain-containing leukocyte protein of 76 kDa: SLP76; Linker for

activation of T cells: LAT; Antigen presenting cells: APC; Granulocyte-macrophage

colony-stimulating factor: GM-CSF; Severe Combined Immunodeficiency: SCID; C-C

chemokine receptor type 7: CCR7; plasmacytoid DC: pDCs; Wild-type: WT; IFNAR

deficient: IFNAR-/-; Renal cell cancer: RCC; PV: Policitemia Vera. ET: Essential

Thrombocitemia. PM: Primary Mielofibrosis




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Abstract

Type I interferons (IFNs-I) are well-known inducers of tumor cell apoptosis and

antiangiogenesis via signaling through a common receptor (IFNAR). IFNAR induces

the JAK-STAT (Janus Activated Kinase-Signal Transducer and Activation of

Transcription) 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 demonstrated that IFNs-I activate directly

other cells of the immune system, such as antigen-presenting dendritic cells (DCs), 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.




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Background

Types of IFN

IFNs were discovered in 1957 by Isaacs and Lindenmann as soluble proteins able to

inhibit virus replication in cell cultures (1). There are three types of IFNs: type I (IFN-

I), Type II (IFN-II) and Type III (IFN-III) (2, 3). IFNs belonging to all IFN classes are

very important for fighting viral infection. In humans, IFNs-I include IFNα subtypes,

IFNβ, IFNε, IFNκ and IFNω (2). From an immunological perspective, IFNα and IFNβ

are the main IFN-I subtypes of interest. Both are produced by almost every cell of the

body in response to viral infection and share important anti-viral properties. The IFNα

family is a multigenic group of highly homologous polypeptides encoded by more than

13 intronless genes in humans. IFNβ, in contrast, exists as a single gene in most species.

IFN-γ is the only IFN-II. This cytokine does not have marked structural homology with

IFNs-I and binds a different cell-surface receptor (IFNGR), also composed of two

different subunits (IFNGR1 and IFNGR2) (2). The recently classified IFN-III consists

of three IFNλ molecules (IFNλ1, IFNλ2 and IFNλ3) and signals through a receptor

complex consisting of IL10R2 (Interleukin-10 receptor 2 subunit) and IL28Rα

(Interleukin 28 receptor alpha subunit) (3). It is well established that IFNs induce the

expression of hundreds of genes, which mediate various biological responses. Some of

these genes are regulated by the three classes of IFNs, whereas others are selectively

regulated by distinct IFNs.

While the role of IFN-II and IFN-III is very important in immune responses, IFN-II has

not shown yet any clinical activity for human cancer treatment (4) and type III IFN is

not currently developed for any indication (3). However, since the discovery of IFNs,




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the use of use of IFN-I in therapy has been very wide. In fact type I IFNs systemic

injections are approved for the treatment of a variety of diseases that include solid and

hematological malignancies, multiple sclerosis and above all chronic viral hepatitis. In

this review we focus on the role of IFNs-I as a direct immunostimulatory agent directly

modifying functions of immune system cells and how can these functions be applied to

better therapeutic strategies.

IFN-I signaling pathways

All IFNs-I signal through a unique heterodimeric receptor (IFNAR) composed of two

subunits, IFNAR1 and IFNAR2, which are expressed in most tissues (5). IFNAR1

knockout mice have not only confirmed the role this subunit plays in mediating the

biological response to IFNs-I but have established the pleiotropic role that these IFNs

play in regulating the host response to viral infections and in adaptive immunity (6-8).

Receptor occupancy rapidly triggers several signaling cascades (Fig. 1) which culminate

in the transcriptional regulation of hundred of IFN-stimulated genes (ISGs). The first

signaling pathway shown to be activated by IFNs-I was the JAK-STAT pathway (9)

(Fig. 1a) which is active in almost all cell types. IFNAR1 is constitutively associated

with tyrosine kinase 2 (TYK2), whereas IFNAR2 is associated with JAK1. Receptor

binding results in transphosphorylation of JAK1 and TYK2. The activated JAKs then

phosphorylate conserved tyrosine residues in the cytoplasmic tails of the IFNAR. These

phosphotyrosyl residues subsequently serve as docking sites for the recruitment of src-

homology 2 (SH2)-containing signaling molecules such as STAT1 and STAT2. Once at

the receptor, the STAT proteins themselves become JAK substrates. STAT1 and

STAT2 are both phosphorylated on a single tyrosine (Y701 for STAT1 and Y690 for

STAT2). Once phosphorylated STATs dimerize and move to the nucleus, where they




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associate with the IFN-regulatory factor 9 (IRF9) to form the ISG factor 3 (ISGF3)

transcription factor complex that binds to IFN-stimulated response elements (ISREs).

Although STAT1 and STAT2 are the most important mediators of the response to

IFNs-I, other STATs, namely STAT3 and STAT5, have been found to be

phosphorylated (activated) by IFNs-I (10-13) (Fig. 1a). STAT4 and STAT6 can also be

activated by IFNα, but this activation seems to be restricted to certain cell types, such as

endothelial or lymphoid cells (14-17). Following phosphorylation, activated STATs

form homo- (STAT1, STAT3, STAT4, STAT5 and STAT6) or heterodimers (STAT1/2,

STAT1/3, STAT1/4, STAT1/5, STAT2/3 and STAT5/6) (10, 11, 18, 19) that

translocate to the nucleus and bind other regulatory sequences known as IFNγ-activated

sites (GAS).

Interestingly, distinct STATs have opposing biological effects. Thus, STAT3 stimulates

growth, while on the contrary STAT1 is a growth inhibitor. On the other hand, IFNs-I-

mediated activation of STAT4 is required for IFNγ production, whereas surprisingly

STAT1 negatively regulates the IFNs-I-dependent induction of IFNγ (20). The relative

abundance of these dimeric transcription factors, which may vary substantially

depending on cell type and activation/differentiation state, is likely to have a major

impact on the overall response to IFNs-I (21-23).

Stimulation of the IFNAR/JAK/STAT1 pathway can also lead to the up-regulation of

the TAM receptor Axl that accumulates at the cell surface where it physically associates

with the IFNAR and usurps the IFNAR-STAT1 pathway by stimulating the

transcription of SOCS (suppressors of cytokine signaling ) (24, 25). The SOCS proteins

extinguish IFN-I signaling in a negative-feedback loop. For instance, SOCS1 directly

interacts with JAKs blocking their catalytic activity whereas SOCS3 seems to inhibit




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JAKs after gaining access by receptor binding. In addition, SOCS proteins interact with

the cellular ubiquitination machinery through the SOCS-box region and may redirect

associated proteins, such as JAKs or IFNAR chains to proteasomal degradation. It has

been shown that SOCS1 knockout mice develop lupus-like disease symptoms (26) and

that SOCS1 silencing enhances the antitumor activity of IFNs-I (27) suggesting that

SOCS are key elements in keeping IFNs-I effects under control.

Alternative signaling pathways and cross-talk with other receptors

There is accumulating evidence that several other signaling elements and cascades are

required for the generation of many of the responses to IFNs-I, such as the CRKL (v-crk

sarcoma virus CT10 oncogene homolog (avian)-like) pathway, the MAPK (mitogen-

activated protein Kinase) pathway, the PI3K (phosphatidylinositol 3-kinase) pathway,

and either the classical or the alternative NF-κB (Nuclear Factor kappa B) cascade (9,

28) (Fig. 1b to 1d). Some of these pathways operate independently of the JAK-STAT,

whereas others cooperate with STATs to tune the response to IFNs-I. Interestingly,

MAPK, PI3K and NF-κB pathways involve immunoreceptor tyrosine-based activation

motives (ITAMs) used by classical immunoreceptors, suggesting a cross-talk among

IFNAR and multiple receptors on immune system cells.

The PI3K and NF-κB pathways. Upon activation, TYK2 and JAK1 phosphorylate

insulin receptor substrate 1 (IRS1) and 2 (IRS2), which provide docking sites for the

PI3K (9). STAT3 acts as an adapter to couple PI3K to the IFNAR1 subunit. PI3K

subsequently activates the serine-threonine kinase AKT that in turn mediates

downstream the activation of mammalian target of rapamycin (MTOR), and the

inactivation of several proteins, such as GSK-3 (Glycogen synthase kinase 3), Cyclin

dependent kinase inhibitors 1A and 1B (CDKN1A and CDKN1B) all of them involved




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in the control of cell division and proliferation. The AKT pathway also results in the

activation IKKβ (IκB kinase beta) that gives rise to NF-κB activation (nuclear factor

kappa-light-chain-enhancer of activated B cells). IFNs-I also activate NF-kB by an

alternative pathway that involves the linkage of Tumor necrosis factor receptor

associated factors (TRAFs) to the activation of the NF-kB-inducing kinase (NIK),

which in turn results in activation of NF-kB2. This alternative pathway is strictly

dependent on the activity of IKKα. In addition, PI3K/AKT can also activate NF-kB

through an activation loop via Protein kinase C zeta (PKCθ). Overall, NF-kB activated

by IFNs-I regulates provides pro-survival signals, enhances the expression of several

guanosine triphosphate (GTP)-binding proteins as well as molecules involved in antigen

processing/presentation.

The MAPK pathway. Activation of JAK results in tyrosine phosphorylation of the

guanine nucleotide exchange factor Vav that leads to downstream activation of Rat

Sarcoma protein (Ras) and Rac1 (ras-related C3 botulinum toxin substrate 1), which

subsequently leads to the activation of several MAPKs: p38, c-Jun N-terminal kinases

(JNK) and Extracellular signal-regulated kinases (ERK) 1 and 2 (9). Vav can also

activate PKCθ leading to the activation of the NF-κB cascade. Several studies have

shown that IFNα-mediated activation of ERK1/2 in T lymphocytes requires proteins

involved in the T cell receptor (TCR) early signaling complex, such as CD45, Lck

(lymphocyte-specific protein tyrosine kinase), Zap70 (Zeta-chain-associated protein

kinase 70), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa) and Vav1, and

possibly LAT (linker for activation of T cells), indicating crosstalk between pathways

(29-31). Moreover, recently it has been shown that TCR-deletion in Jurkat cells

abrogated IFNAR-stimulated MAPK activity, whereas the canonical JAK-STAT

pathway remained unaffected (31). A hypothetical pathway that involves all these




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molecules and results in Vav activation is depicted in Figure 1d. The different MAPKs

activate different sets of transcription factors. Importantly, p38 functions are essential

for the antiviral and antileukemic properties of IFNs-I, plays a role in the

hematopoietic-suppressive signals and is required for the growth-inhibitory effects of

IFNs-I observed in certain lymphocyte cultures. IFNs-I-activated ERK cascades

regulate cellular growth, differentiation and serine phosphorylation of STAT1. The

search for biochemical signals to explain the effects of type I IFN on immune system

cells is far from being over.

Effects of IFNs-Is on cells of the immune system

It has long been established that the main mechanism accounting for the efficacy of the

IFN-I-based therapies was due to their direct effect on malignant or virus-infected cells.

In fact, IFNs-I directly inhibit the proliferation of tumor and virus-infected cells and

increase MHC class I expression, enhancing antigen recognition. Moreover, IFNs-I

repress oncogene expression and induce that of tumor suppressor genes, which may

contribute to the inhibitory effects of IFN-I on malignant cells in conjunction with the

antiangionic effects.

IFNs-I have also proved to be involved in immune system regulation (7, 8, 32). IFNs-I

exert their effects on immune cells either directly, through IFNAR triggering or

indirectly by the induction of chemokines, that allow the recruitment of immune cells to

the site of infection; the secretion of a second wave of cytokines, that could further

regulate cell numbers and activities (as for example IL-15, which plays a critical role in

proliferation and maintenance of NK cells and memory CD8 T cells) (33, 34); or

through the stimulation of other cell types critical for the activation of certain immune

cells, such as DC for the activation of naïve T cells.




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One of the earliest described immunoregulatory functions of IFNs-I was their ability to

regulate NK functions (32). IFNs-I enhance the ability of NK cells to kill target cells

and to produce IFNγ by indirect and direct mechanisms (33, 35, 36). Furthermore,

IFNs-I promote the accumulation/survival of proliferating NK cells by the IFN-

I/STAT1-dependent induction of IL-15 (33). IFNs-I also enhance the production or

secretion of other cytokines by the NK cell through the autocrine IFNγ loop (37).

IFNs-I also affect monocyte/macrophage function and differentiation. Thus, IFNs-I

markedly support the differentiation of monocytes into DC with high capacity for Ag

presentation, stimulate macrophage antibody-dependent cytotoxicity and positively or

negatively regulate the production of various cytokines (e.g. Tumor Necrosis Factor, IL-

1, IL-6, IL-8, IL-12 and IL-18) by macrophages (38). In addition, autocrine IFN-I is

required for the enhancement of macrophage phagocytosis by Macrophage colony-

stimulating factor and IL-4 (39) and for the Lipopolysaccharide-, virus- and IFNγ-

induced oxidative burst through the generation of Nitric oxide synthase 2.

The main function of DC is to uptake and process antigens for presentation to T cells.

DCs are the unique cell type able to prime naïve T cells and therefore are critical

antigen presenting cells (APC). IFNs-I have multiple effects on DCs, affecting their

differentiation, maturation and migration. Thus, human monocytes cultured in

Granulocyte-macrophage colony-stimulating factor (GM-CSF)+IFNα/β, rather than the

more commonly used combination of GMCSF+IL-4, differentiate more rapidly into

DCs, exhibiting the phenotype of partially mature DCs while showing a strong

capability to induce a primary human antibody response and CTL expansion when

pulsed with antigen and injected into humanized Severe Combined Immunodeficiency

(SCID) mice (40-42).




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In vitro treatment of immature conventional DCs with IFNα/β has been shown to up-

regulate surface expression of MHC Class I, Class II, CD40, CD80, CD86 and CD83

molecules in the human system (43-45), associated with a heightened capacity to induce

CD8 T-cell responses. Accordingly, it has been observed in mice that IFNα acting on

DC plays a key role in achieving efficient cross-priming of antigen-specific CD8 T

lymphocytes (7, 8, 32). IFNs-I also affect the ability of DC to secrete IL-12p70. Low

concentrations of IFNs-I are essential for the optimal production of IL-12p70 (46),

whereas higher levels of type I IFN suppress IL-12p40 expression, thus dampening IL-

12p70 production (47).

A key requisite to initiate the adaptive immune response is the arrival of professional

APC from infected tissue into lymph nodes. It has been shown that human DC

differentiated from monocytes in the presence of IFNα exhibited up-regulation of CC

chemokine receptor type 7 (CCR7) correlating with an enhanced chemotactic response

in vitro to CCL19 (CCR7 natural ligand) and with strong migratory behavior in SCID

mice (48). Interestingly, experiments in mice have also shown that IFNα/β is required

for plasmacytoid DC (pDCs) to migrate from the marginal zone into the T cell area

where they form clusters (49). We have also recently shown that IFNs-I enhance the

adhesion and extravasations of DC across inflamed lymphatic endothelium in a

Lymphocyte function-associated antigen 1 (LFA-1)- and Very Late Antigen-4 (VLA4)-

dependent fashion (50). IFNs-I may also favor the encounter between DC and specific

lymphocytes in lymph nodes by promoting lymphocyte trapping in lymph nodes upon

down-modulation of Sphingosine 1-phosphate (51).

IFNs-I have also been shown to potently enhance the primary antibody response to

soluble antigen, stimulating the production of all subtypes of IgG, and inducing long-




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lived antibody production and immunological memory (8). Direct effects of IFNα on

DC (8), B cells (52, 53) and CD4 T cells (53) all contribute to its adjuvant activities on

humoral immune responses. Interestingly, a direct effect of IFNs-I on B cells seems to

be crucial for the development of local humoral responses against viruses (54).

As mentioned above, CD4 T cells are direct targets of IFN-I in the enhancement

of antibody responses (53). In humans, it has also been described that direct effects of

IFN-I on naive CD4 T cells favor their differentiation into IFNγ secreting Th1-like T

cells (55).

Several studies suggested that IFNs-I might exert a direct effect on CD8 T cells.

Thus, it was reported that IFNs-I promote IFNγ production by CD8 T cells in a STAT4-

dependent manner (56) and promote survival of CD8 T cells from Wild-type (WT) but

not IFNAR deficient (IFNAR-/-) mice (57). The definitive report showing that CD8 T

cells represent direct targets of IFN-I-mediated stimulation in vivo came from Kolumam

et al (58). By adoptively transferring virus-specific naive CD8 T cells from IFNAR-/- or

IFNAR–sufficient mice into normal IFNAR WT hosts, IFNs-I were shown to act

directly on murine CD8 T cells allowing their clonal expansion and memory

differentiation. Subsequent studies confirmed this work (59-61). Elegant experiments in

mice by the group of Mescher (62) have shown that, in addition to signals via TCR

(signal-1) and CD28 (signal-2), naïve CD8 T cells required a third signal to differentiate

into effector cells. cDNA microarray analyses showed that IFNα could regulate critical

genes involved in CTL functions (63), providing evidence that IFNα promoted

activation and differentiation of CD8 T cells by sustaining the expression of T-bet and

Eomes through chromatin remodeling. Recently we have shown that IFNα provides a

strong and direct signal to human CD8 T cells thereby resulting in up-regulation of




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critical genes for cytotoxic T cell activity (cytolysis and IFNγ-secretion) and for the

production of chemokines that would co-attract other effector lymphocytes. IFNα was

absolutely critical in the case human naïve CD8 T cells for effector function acquisition

(64). In many instances T cells may sense IFN-I before the cognate antigen is actually

presented to them. As a result of a prexposure to IFN-I, CD8 T cells become

preactivated and acquire much faster effector functions upon antigen recognition (65).

This may reflect the up-regulation by IFN-I of several mRNAs in antigen-naive CD8 T

lymphocytes albeit without changes in the expression of the corresponding protein

unless antigen-specific activation ensues (64).




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Clinical-Translational Advances

The contracting current indications of IFNα as an anticancer agent

IFNα has received approval for treatment of several neoplastic diseases (66). The most

frequent indications for IFNα are chronic viral hepatitis. For the treatment of these

infectious conditions stabilized forms of IFNα have been created by directed

conjugation of polyethylene glycol, which by increasing molecular weight retards renal

clearance and thereby extends plasma concentrations with sustained receptor occupancy

(67). However, PEG-conjugated IFN is not widely used in oncology in the absence of

comparative clinical studies with the unconjugated form.

In oncology the main indication of IFNα is for patients with resected stage II and III

melanoma, in whom IFNα prolongs disease-free survival and shows a trend towards

increased overall survival (68). However, the advent of CTLA-4 antagonist antibodies

will very likely displace the use of IFNα for melanoma therapy (69).

Table I shows the spectrum of neoplastic diseases in which IFNα has been used. As it

can be seen its role in cancer therapy is steadily decreasing because of its limited

efficacy and the advent of new, more effective and/or safer treatments.

Future perspectives

IFNs-I are powerful tools to directly and indirectly modulate the functions of the

immune system. Evolution has shaped these cytokines as a key sign of alarm in case of

viral infection. The type of immune response that we would like to induce and sustain

against cancer antigens is identical to the one we commonly observe upon acute viral




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infections. Obviously, the effects of IFN-I are integrated with many other key

molecules that need to act in a concerted fashion.

As described in table I, side effects of systemic long term treatments and lack of

sufficiently high efficacy have dampened the interest of IFNα for clinical use in

oncology. However, we believe that IFNα is likely to be more efficacious when acting

locally and intermittently at the malignant tissue and tumor draining lymph node than

when administered to achieve systemic bioavailability. Local delivery can be achieved

with pharmaceutical formulations and gene therapy approaches (70). In our opinion, the

clinical use of recombinant IFNα as a vaccine adjuvant should be reconsidered and new

investigations carried out with an eye to clinical applications (71).

The reasons to use intermittent delivery arise from the observations indicating

that IFN-I turn on the mechanisms that desensitize signaling (i.e because of SOCS1

induction). Intermittency at an optimized pace may help to avoid these negative feed

back mechanisms in the responding immune cells. These ideas contrast with stabilized

formulations such as the widely used polyethylene glycol-conjugated IFNα and fusions

proteins with albumin that extend the half-life of the cytokine.

It is of much interest that a gene expression signature induced by IFN-I in

melanoma lesions predicts benefit from vaccines and may have a key role in recruiting

effector T cells to tumors by inducing chemokine genes (72). IFNα has been recently

used in promising combinatorial immunotherapy approaches alongside DC vaccination

for glioblastoma, precisely to promote T cell infiltration and DC activation (H. Okada. J

Clin Oncol in press). In this regard, using inducers of endogenous type I IFN instead of

the recombinant cytokines may have advantages, since substances such as the viral




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RNA analog PolyI:C will also elicit other cytokines that may act in concert with type I

IFNs to enhance the antitumor immune response.

Creative combination strategies building on the knowledge that IFNα effects on

the immune system are likely to improve its therapeutic profile against malignant

diseases. For instance, combinations with immunostimulatory monoclonal antibodies,

IL-15, IL-12 and IL-21 may hold much promise. Indeed, intratumoral release of mouse

IFNα along with systemic treatment with anti-CD137 mAb results in synergistic

therapeutic effects (73). Macrophages homing to tumor transduced to express IFNα

exert therapeutic effects without the systemic side effects (74). These studies suggest

that local delivery as opposed to systemic administration should be tested. It is of great

importance the notion that the chemokines induced by IFN-I (i.e CXCL9, CXCL10)

attract activated lymphocytes thereby calling more immune cells to infiltrate the

malignant focus.

When rethinking IFN-I for cancer, we must think about reprogramming the

tumor microenvironment frequently reach in immunocompetent cells. Local IFN-I

release may help to turn some of the macroscopic tumor lesions into immunogenic

vaccines that can be concomitantly treated to undergo immunogenic cell death with

irradiation or chemotherapy. Factors such as CpG oligonucleotides or poly I:C that

induce the release of endogenous IFNs-I at a given tumor lesion (75) are promising in

this regard. These agents may constitute an advantageous alternative because these

agents also induce an additional array of proinflammatory mediators acting in synergy

with IFNs-I.

In conclusion, the notion of key direct effects of IFNs-I on immune system cells

and detailed knowledge on the elicited signaling pathways should help biomarker




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discovery to identify the small number of patients who could actually benefit from

current treatments. More importantly, these ideas might change the way we use IFNα

for cancer therapy suggesting new strategies and combinations with other agents.

Acknowledgments:

We are grateful for continuous scientific collaboration on type I IFNs with Jesús Prieto,

Esther Larrea, Pilar Civiera, Iranzu Gonzalez and Juan Ruiz. Finacial support comes

from MEC/MCI (SAF2005-03131, SAF2008-03294 and TRA2009_0030),

Departamento de Educación y Departamento de Salud (Beca Ortiz de Landázuri) del

Gobierno de Navarra. Redes temáticas de investigación cooperativa RETIC

(RD06/0020/0065), Fondo de investigación sanitaria (FIS PI060932), European

commission VII famework program (ENCITE), Immunonet SUDOE. Fundación Mutua

Madrileña, and “UTE for project FIMA”. S.H.-S. was supported by AECC and by MCI

(RYC-2007-00928).

Conflict of interest:

I.M., A.R. and S.H.-S. receive laboratory reagents and research funding from Digna

Biotech. I.M. has acted as a consultant for Bristol Myers-Squibb and Pfizer Inc.




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Figure 1. Signaling Pathways activated by the IFN-I receptor. Schematic

representation of signaling pathways downstream of the IFNAR, emphasizing

crosstalk with different signaling cascades that depends on the identity and

activation status of the cell responding to type I IFN. (A) JAK-STAT-signally

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-κB pathways. Phosphorylation

of TYK2 and JAK1 results in activation of PI3K and AKT that in turn mediates

downstream activation of MTOR, inactivation of GSK-3, CDKN1A and CDKN1B

and activation of IKKβ that result in activation of NF-κB. 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 PKCθ. (D) MAPK Pathway. Activation of JAK results in tyrosine

phosphorylation of Vav that 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).

(76) (77) (78) (79) (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) (91) (92)

(93)




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20

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22

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Table 1. Contracting indications of IFNα for malignant conditions.

PV: Policitemia Vera; ET: Essential Thrombocitemia; PM: Primary Mielofibrosis

Indication Stage Comment Standard current therapy Reference Melanoma

II/III Prolongs disease free survival and shows a trend towards increased overall survival.

IFNα is the standard option. Chemotherapy. The advent of CTLA-4 antagonist antibody will displace the use of IFNα for melanoma inmunotherapy.

(68)

(76, 77)

IV

Combination with IL2: Increase response rates but no survival improvement.

Renal cell Cancer (RCC)

II/III Two Phase III trials showed negative results. Novel targeted agents such as sunitinib, temsirolimus, sorafenib or everolimus have decreased to a minimum the use of IFNα in both resectable and advanced RCC.

(78, 79)

IV Widely studied as a single agent or in combination with IL-2, chemotherapy or both: Modest or negative results.

(80)

Hairy cell Leukemia First treatment that showed clinical benefit in this disease. Recommended, in low dose, as a therapeutic option for maintenance therapy and as an alternative for frail patients.

Purine analogs are now considered standard treatment for this disease.

(81, 82)

Multiple Mieloma

Extensively studied as maintenance treatment, as single treatment or in combination with chemotherapy: Slight efficacy.

Largely replaced by newer and more effective agents such as thalidomide, lenalidomide or bortezomib.

(83)

Chronic Myeloproliferative Syndromes (CMS)

PV Optional treatment for cytoreduction (94,6% complete responses). The mainstay of therapy remains repeated phlebotomy.

(84-86)

ET Treatment of choice in women with childbearing potential and optional treatment in young patients resistant to hidroxiurea. Hidroxiurea. Anagrelide.

(85)

PM Cytoreduction therapy. Hidroxiurea. Thalidomide. (76)

Chronic Myeloid Leukemia (CML)

First-line therapy in combination with cytarabine: 71% clinical response rate and 39% major cytogenetic response rate. In comparison with chemotherapy IFNα has been showed superior.

Imatinib, in a phase III trial, have been superior in tolerability, complete hematologic and cytogenetic response rates and progression-free survival. In imatinib resistance, IFNα has been precluded by the appearance of dasatinib.

(87-91)

Hemangioma Complicate hemangiomas, that do not respond to steroids. Rate regression in 58% of the patients.

Steroids. (92)

AIDS related Kaposi sarcoma

Evidence for clinical activity. Liposomal anthracyclines are preferred as the first treatment option.

(93)

Research.

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ownloaded from

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© 2011 American Association for Cancer Research

Tyk2

IFN /(A) JAK-STAT

STAT

STAT STAT

STAT

STAT3

STAT3

C3G

IRF9

IRF9

ISG15, IP-10,IRF-7, PKR,2’-5’-OAS,etc

IRF-1, IRF-2,IRF-8, IRF-9,etc

Regulation of expression of

Survival signals

Apoptosis Cellular growthDifferentiationSer phosphorylationof STAT1

c-MycSAP-1P53SP1SMADs

NFATEtsElk-1MEF2Stat1/3

JunElk-1NFATATF2

Ras

Raf1

MEK1/2

ERK1/2JNKp38

Rac1Rap1

ProliferationDifferentiationSurvival....

mTOR

GSK-3/CDKN1A/CDKN1B

PKCIKKIKK

NF B

MEF2MAPKAPRsk

FosElk-1Sap-1

ISG15Histone acetylationAntiviral effect

Hematopoietic suppressive signals

STAT1

STAT5

STAT5

CRKL

CRKL

CRKL

STAT2

STAT1

STAT2

VAV

JAK1 JAK1 JAK1 JAK1

VAV

VAV

STAT1, STAT3STAT4, STAT5STAT6

STAT1/2,3,4 or 5STAT2/3STAT5/6

STATSTAT

IFN

AR

1IF

NA

R2

IFN /(B) CRK

IFN / IFN / IFN /

Type I IFNs signaling pathways

(C) PI3K and NF BTCR complex

CD45

CD4/8

(D) MAPK

IFN

AR

1IF

NA

R2

Homodimers

STAT STAT

STAT STAT

Homodimers

Heterodimers

Heterodimers

ISRE GAS GAS

Regulatedgenes orfunctions

NF B Rel ... ... ...

Tyk2IF

NA

R1

IFN

AR

2

IFN

AR

1IF

NA

R2

IFN

AR

1IF

NA

R2

Tyk2

NIK

Tyk2

RS1/2PI3K

AKT

Zap70Lck

LA

TSLP76TRAFSTAT

5C3G




Published OnlineFirst March 3, 2011.Clin Cancer Res Sandra Hervas-Stubbs, Jose Luis Perez-Gracia, Ana Rouzaut, et al. Direct Effects of type I IFNs on cells of the immune system

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