Severe Crohn's Disease Semapimod Induces Clinical Remission in Specific Inhibition of C-Raf Activity By

Specific Inhibition of c-Raf Activity by Semapimod Induces Clinical Remission in Severe Crohn's Disease

This information is current as Mark Löwenberg, Auke Verhaar, Bernt van den Blink, Fibo of September 24, 2021. ten Kate, Sander van Deventer, Maikel Peppelenbosch and Daniel Hommes J Immunol 2005; 175:2293-2300; ; doi: 10.4049/jimmunol.175.4.2293

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Speciﬁc Inhibition of c-Raf Activity by Semapimod Induces Clinical Remission in Severe Crohn’s Disease1

Mark Lo¨wenberg,2*‡ Auke Verhaar,* Bernt van den Blink,* Fibo ten Kate,† Sander van Deventer,* Maikel Peppelenbosch,§ and Daniel Hommes‡

There is a substantial need for novel treatment strategies in Crohn’s disease (CD), a chronic relapsing inflammatory disease of the gut. In an earlier study, we reported clinical efficacy of a 2-wk treatment with semapimod (CNI-1493) in 12 patients with therapy resistant CD. The aim of this study was to identify the cellular target underlying semapimod action. In vitro experiments with murine macrophages showed impaired MAPK signaling and decreased cytokine production due to semapimod treatment. In vitro kinase assays revealed c-Raf as a direct molecular target of semapimod, and semapimod did not affect b-Raf enzymatic activity. demonstrated increased expression (6 ؍ Immunohistochemistry performed on paired colon biopsies obtained from CD patients (n of phospho-MEK, the substrate of Raf. Strikingly, phospho-MEK levels were significantly decreased in patients with a good Downloaded from clinical response to semapimod, but no decrease in phospho-MEK expression was observed in a clinically nonresponsive patient. In conclusion, this study identifies c-Raf as a molecular target of semapimod action and suggests that decreased c-Raf activity correlates with clinical benefit in CD. Our observations indicate that c-Raf inhibitors are prime candidates for the treatment of CD. The Journal of Immunology, 2005, 175: 2293–2300.

nhibitors of intracellular signaling pathways have proven ef- small molecule semapimod resulted in a reduction of disease ac- http://www.jimmunol.org/ fective in a wide range of experimental inflammatory disor- tivity and induction of clinical remissions (14). Although it has I ders, including experimental colitis (1). Small molecules tar- been demonstrated that semapimod interferes with the phosphor- geting these signaling cascades are generally considered as a ylation of both p38 and JNK (14), the exact underlying molecular promising novel strategy for the clinical management of inflam- mechanism of semapimod action remains to be characterized. The matory bowel diseases (i.e., Crohn’s disease (CD)3 and ulcerative identification of the molecular target of semapimod has important colitis). In particular, pharmaceutical intervention of the MAPK clinical relevance because it may prompt synthesis of a novel class pathways of intracellular signaling mediators attracts widespread of anti-inflammatory compounds. In this study we have identified interest (2–6). Three major MAPK cascades have been identified: macrophages as the target cells of semapimod action, and we char- ERK, JNK, and p38 MAPK, and these pathways are critically in- acterized c-Raf as the molecular target. Reduced expression of by guest on September 24, 2021 volved in inflammatory pathology, including CD (6–8). Selective phospho-MEK, a downstream target of c-Raf, in colon biopsies MAPK inhibitors targeting the p38 MAPK, ERK, and JNK path- correlated with clinical benefit in semapimod-treated CD patients. way demonstrated anti-inflammatory effects in preclinical models In contrast, no reduced phospho-MEK was observed in mucosal (1, 9–13). Despite the fact that the impact of MAPK pathways on biopsies obtained from a nonresponder. These results indicate that inflammatory pathology is profound, the molecular details of these c-Raf activity is a critical mediator of disease progression in CD, signaling cascades in the pathogenesis of inflammatory disorders and identify c-Raf as a novel therapeutic target for the clinical and their possible therapeutic value remain to be elucidated. In management of CD. view of the redundancy of MAPK pathways and the extensive cross-talk between these and other routes of signal transduction Materials and Methods (e.g., NF-␬B), such information is of great importance. We have Abs and reagents reported that treatment of therapy resistant CD patients with the Phospho-specific Abs directed against p38Thr180/Tyr182, ERK1/2Thr202/Tyr204, MEK1/2Ser217/221, c-RafSer338, stress-activated protein kinase/JNKThr183/Tyr185, p21-activated protein kinase (PAK)1/2Thr423/402, SEK1/MAPK kinase *Laboratory of Experimental Internal Medicine, †Department of Pathology, and ‡De- (MKK)4Thr261, MKK3/pMKK6Ser189/207, as well as Abs specific for MKK4, partment of Gastroenterology and Hepatology, Academic Medical Center, Amster- MKK3, and PAK were purchased from Cell Signaling Technology. Abs rec- dam, The Netherlands; and §Department of Cell Biology, University of Groningen, ognizing p38, ERK, JNK, MEK, b-Raf, c-Raf, and phospho-JNKThr183/Tyr185 Groningen, The Netherlands were from Santa Cruz Biotechnology. HRP-conjugated goat anti-rabbit, goat Received for publication January 25, 2005. Accepted for publication May 18, 2005. anti-mouse, and rabbit anti-goat were from DakoCytomation, and semapimod The costs of publication of this article were defrayed in part by the payment of page (CNI-1493) was acquired from Cytokine PharmaSciences (batch date 3/13/ charges. This article must therefore be hereby marked advertisement in accordance 2004; lot no. 08610302). The anti-CD68 mAb was from DakoCytomation, and with 18 U.S.C. Section 1734 solely to indicate this fact. anti-CD14 mAb was obtained from BD Biosciences. Anti-human CD3 (CD3⑀ mouse) was kindly provided by Dr. A. te Velde (Academic Medical Center, 1 This work is supported in part by grants from The Netherlands Organization for Health Research and Development (to D.H.), and from the Dutch Digestive Disease Amsterdam, The Netherlands). Anti-CD28 was from Sanquin. The c-Raf and Foundation (to M.P.). This study was performed with a grant kindly provided by b-Raf kinase kits were obtained from Upstate Biotechnology. Cytokine PharmaSciences (King of Prussia, PA). CD4 purification and cell sorting 2 Address correspondence and reprint requests to Dr. Mark Lo¨wenberg, Laboratory of Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, NL- PBMC were isolated from whole blood of healthy volunteers by Ficoll- 1105 AZ Amsterdam, The Netherlands. E-mail address: [email protected] Isopaque density gradient centrifugation (Amersham Biosciences). The 3 Abbreviations used in this paper: CD, Crohn’s disease; DC, dendritic cell; CRP, monocytes present in the PBMC pellet were removed by an adherence C-reactive protein; CBA, cytokine bead array; CDAI, Crohn’s Disease Activity In- procedure: cells were plated out in 6-well plates (Cellstar; Greiner Bio- dex; MKK, MAPK kinase; PAK, p21-activated protein kinase. One) at a final concentration of 5 ϫ 106 cells/well for 1.5 h at 37°C, and

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 2294 SEMAPIMOD-INDUCED INHIBITION OF c-Raf ACTIVITY subsequently, nonadherent cells were harvested for magnetic cell sorting. Protifar in TBST (0.05 M Tris, 150 mM NaCl, and 0.05% Tween 20). CD4ϩ T cells were purified by depletion of non-CD4ϩ T cells (negative Primary and secondary HRP-conjugated Abs were diluted in 1% Protifar in selection) using the MACS system. Non-CD4ϩ cells were indirectly mag- TBST, and proteins were visualized using the Lumi-Light substrate netically labeled with a mix of biotin-conjugated mAbs (against CD8, (Roche). Blots were incubated in stripping buffer (62.5 mM Tris-HCl (pH CD14, CD16, CD19, CD36, CD56, CD123, TCR ␥␦, and glycophorin A) 6.8), 100 mM 2-ME, and 2% SDS) for1hat50°C and subsequently bound to MicroBeads conjugated to a monoclonal anti-biotin Ab, as sec- reprobed with appropriate Abs to evaluate for equal loading. In addition, T ondary labeling agent (Miltenyi Biotec). The magnetically labeled non- cells (overnight cultured in 6-well plates, 3 ϫ 106 cells/well) pretreated CD4ϩ T cells were depleted by retaining them on a MACS Column in the with semapimod (1 h, 0.1 and 1 ␮M) and subsequently activated with magnetic field of the autoMACS Separator (Miltenyi Biotec), whereas the anti-CD3/anti-CD28 Abs (15 min) were analyzed on Western blot using ϩ unlabeled fraction of CD4 Th cells passed through the column. The sam- phosphospecific Abs. ple purity was assessed by FACS (BD Biosciences) with PE-conjugated CD4 and FITC-conjugated CD3 mAbs (BD Biosciences) (purity Ͼ95% ϩ ϩ CD3 CD4 ; data not shown). Raf in vitro kinase assays Generation of dendritic cells (DC) Raf in vitro kinase assays were used according to the instructions of the DC generation from PBMC (obtained form healthy volunteers) was per- manufacturer (Upstate Biotechnology). Truncated constitutively active Raf (b-Raf and c-Raf) was diluted in a Mg/ATP mixture and reaction formed as previously described (15, 16). Briefly, PBMC were resuspended ␮ in Adoptive Immunotherapy Media (Invitrogen Life Technologies), and buffer, and incubated on ice with semapimod (1 M) for 5 and 10 min. allowed to adhere to 6-well plates (Cellstar; Greiner Bio-One). After2hat Next, recombinant inactive MEK was added and in vitro kinase assays 37°C, nonadherent cells were removed and the adherent cells were cultured were performed at 30°C for 20 min. Active Raf together with MEK and in medium supplemented with 50 ng/ml GM-CSF and 1000 U/ml IL-4. Raf without MEK served as a positive and negative control, respec- Next, monocytes were incubated for 6 days in X-VIVO 15 medium (Bio- tively. Samples were dissolved in sample buffer, incubated at 95°C for 5 min, and analyzed on Western blot using an anti-phospho- Downloaded from Whittaker) supplemented with 1000 U/ml GM-CSF (Berlex) and 1000 Ser218/222 Ser222/226 U/ml IL-4 (R&D Systems). The immature DC were stimulated at day 6 in MEK /MEK2 Ab. X-VIVO 15 medium supplemented with a cytokine mix containing TNF-␣ ␮ ␤ (10 ng/ml), PGE2 (1 g/ml), IL-1 (10 ng/ml), IL-6 (150 ng/ml), GM-CSF (800 U/ml), and IL-4 (500 U/ml). After 24 h, mature DC were harvested for Immunohistochemistry phenotyping using a panel of mAbs and analyzed on a FACScan with To assess the amount of active MEK in the intestinal mucosa, screening CellQuest software (BD Biosciences), as previously described (17). and week 4 colon specimens were obtained from most affected regions of http://www.jimmunol.org/ Cell culture inflammation of CD patients (n ϭ 6) who participated in the semapimod (CNI-1493) study (14). Biopsies were analyzed for phospho-MEK expres- 4/4 macrophages (murine), which are phenotypically and functionally not sion. Paraffin sections (4 ␮m) were dewaxed and rehydrated in graded different from primary isolated mature macrophages (18, 19), were cultured alcohols, and endogenous peroxidase activity was quenched with 1.5% in RPMI 1640 (Invitrogen Life Technologies), supplemented with 10% H2O2 in methanol (15 min, room temperature). Ag retrieval was performed heat-inactivated FCS, 2 mM L-glutamine, and penicillin-streptomycin ϩ by heating for 10 min at 100°C in 0.01 M sodium citrate. After washing (“complete”) in a humidified 5% CO2 environment at 37°C. Human CD4 (PBS), nonspecific staining was reduced by a blocking step with 10 mM T cells were grown in IMDM (Invitrogen Life Technologies), supple- Tris, 5 mM EDTA, 0.15 M NaCl, 0.25% gelatin, 0.05% (v/v) Tween 20 mented with 10% FCS, 2 mM L-glutamine, and penicillin-streptomycin (pH 8.0) for 20 min at room temperature. Subsequently, slides were washed Ser218/222 (complete) in a humidified 5% CO2 environment at 37°C. and incubated overnight (at 4°C) with an anti-phospho-MEK / Ser222/226 MTT viability assay MEK2 Ab diluted in 1% BSA 0.1% Triton X-100. Slides were by guest on September 24, 2021 incubated for 20 min with a post-Ab blocking solution for PowerVision The cytotoxic effect of semapimod was studied in macrophages, which (ImmunoLogic), followed by a 30 min incubation with poly-HRP-GAM/ were incubated overnight with increasing concentrations of semapimod R/R IgG (ImmunoLogic). Peroxidase activity was detected using diami- (0.01, 0.1, 1, 10, and 100 ␮M diluted in medium) with or without LPS (100 nobenzidine (Fast DAB; Sigma-Aldrich) in 0.05 M Tris (pH 7.4). Sections ng/ml). Cell viability was assessed by MTT colorimetric assay. After over- were briefly counterstained with hematoxylin (Mayer’s; Fluka) when ap- night incubation, 0.5 mg/ml MTT was added to the medium for 1–2 h at propriate, dehydrated in graded alcohols, and mounted with Pertex (His-

37°C, and subsequently isopropanol/0.04 N HCl was added. The OD560 tolab Products) under coverslips. Controls consisted of omitting the pri- was determined using an ELISA plate reader (Bio-Rad). Treatment with mary and secondary Ab and use of an appropriate Ig control (data not semapimod concentrations Յ 1 ␮M did not affect cell viability. A semapi- shown). mod-induced cytotoxic effect was observed at semapimod concentrations Ͼ 1 ␮M (10 and 100 ␮M; data not shown). Statistical analysis Cytokine bead array (CBA) ϩ Quantitative confirmation came from experiments in which the number of Macrophages and CD4 T cells were pretreated for 1 h with various con- phospho-MEK-positive cells was counted in sections in a blinded fashion. centrations of semapimod and cultured up to 24 h with either LPS (100 ϫ ␮ Two pictures of each section were taken at 200 magnification, and pos- ng/ml) or anti-CD3 (immobilized on plastic) and anti-CD28 (3 g/ml, sol- itive cells were counted, blind to treatment and day of endoscopy in each uble) Abs, respectively. Furthermore, mature DC were pretreated for 1 h ␮ microscope field with the use of an image analysis program (EFM Soft- with 0.1 and 1 M semapimod. Medium was removed, and cells were ware). Pictures appeared randomly on a computer monitor and all intensely cultured for 24 h in fresh medium containing CD40L transfected J558 cells staining cells were marked positive by an observer, counted, and stored by (1:1). The CD40L transfected mouse plasmacytoma cell line (J558), was a the image analysis program for later data analysis. Statistical analysis was kind gift from Dr. P. Lane (University of Birmingham, Birmingham, U.K.) performed by use of the Wilcoxon test, and a value of p Ͻ 0.05 was (20). Cytokine levels were analyzed in supernatants of macrophages, DC, considered as statistically significant. and T cells by CBA (BD Biosciences) using a flow cytometer (BD Bio- sciences), according to routine procedure. Western blot analysis Results MAPK signaling pathways were studied on Western blot using phos- Semapimod does not affect MAPK signaling cascades in T cells phospecific Abs against a variety of MAPK signal transduction molecules. Macrophages were seeded in 6-well plates at a final concentration of 1–2 ϫ It has been previously reported that T cell cytokine production is 106 cells/well and grown overnight. Cells were pretreated for 1 h with 0.1 not influenced by semapimod (21), and this was confirmed in our and 1 ␮M semapimod and subsequently stimulated with LPS (100 ng/ml) laboratory (data not shown). ERK, JNK, and p38 MAPK signal for 15 min. After washing with PBS, cells were harvested in sample buffer transduction pathways were activated in T cells stimulated with (150 mM Tris-HCl, 6% SDS, 3% 2-ME, 20% glycerol, and 1 mg of bro- mophenol blue (pH 6.8)), and whole cell lysates were loaded on 10% anti-CD3, anti-CD28 Abs, and this was not affected by incubation SDS-PAGE and subsequently transferred to a polyvinylidene difluoride with semapimod (Fig. 1). Thus, these findings indicate that T cells membrane (Immobilon-P; Millipore). Membranes were blocked with 1% are no direct target of semapimod action. The Journal of Immunology 2295

Semapimod inhibits cytokine production in macrophages To determine the usefulness of in vitro stimulation of macrophages for studying the underlying molecular mechanism of semapimod action, the effect of semapimod on cytokine production in macrophages was investigated. Semapimod treatment resulted in a dose responsive reduction of LPS-induced TNF-␣, IL-1␤, and IL-6 protein levels (Fig. 3). Decreased cytokine production observed upon treatment with 0.01, 0.1, and 1 ␮M semapimod was not a result of reduced cell viability, as MTT colorimetric assays revealed sig- niﬁcant cytotoxicity of semapimod only at concentrations of 10 and 100 ␮M (data not shown). These observations conﬁrm that semapimod effectively blocks cytokine synthesis in macrophages (27–29) and indicate that incubation of macrophages with semapimod concentrations of 0.1 and 1 ␮M constitute an appropriate

FIGURE 1. Semapimod does not inﬂuence phosphorylation of MAPKs in T cells. The effect of semapimod on ERK, JNK, and p38 MAPK in activated T cells was analyzed by Western blotting. Cells were pretreated with semapimod for 1 h and subsequently stimulated for 15 min with (ϩ) Downloaded from or without (Ϫ) anti-CD3 and anti-CD28 Abs. Semapimod did not affect phosphorylation of ERK, JNK, or p38 MAPK in activated cells. To test for equal loading, blots were reprobed with appropriate Abs. Western blots represent three independent experiments, and duplo conditions are shown. http://www.jimmunol.org/

Semapimod does not inﬂuence IL-12 cytokine production in activated mature DC Recently, it has been reported that semapimod interferes with DC maturation (22), which prompted us to study the effect of semapimod on mature DC. The effect of semapimod on IL-12 cytokine production was studied in CD40L-activated mature DC because it is generally accepted that this is an important Th1 differentiation by guest on September 24, 2021 mechanism that is relevant for CD (23–26). Our data indicate that semapimod does not interfere with IL-12 cytokine production in activated mature DC, suggesting that this compound does not in- ﬂuence the capacity of DC to induce a Th1-type response (Fig. 2).

FIGURE 2. Semapimod does not affect IL-12 cytokine production in FIGURE 3. Semapimod blocks TNF-␣, IL-1␤, and IL-6 cytokine pro- activated mature DC. Mature DC were incubated for 1 h with semapimod duction in activated macrophages. Cells were cultured overnight with in- and subsequently cultured for 24 h in the presence of CD40L-overexpress- creasing semapimod concentrations in the absence (छ) or presence (ࡗ)of ing cells. IL-12 cytokine levels were analyzed in supernatants by CBA, LPS, and supernatants were analyzed by CBA. A decrease in cytokine demonstrating increased IL-12 cytokine levels in activated mature DC. levels observed upon treatment with 0.01, 0.1, and 1 ␮M semapimod was Semapimod treatment did not interfere with IL-12 cytokine production. not a result of reduced cell viability, as MTT colorimetric assays revealed IL-12 cytokine levels in supernatants of control cells were not measurable significant toxicity of semapimod only at concentration of 10 and 100 ␮M. and therefore error bars are not appropriate. Results are expressed as the These data confirm findings reported previously. Results are expressed as mean Ϯ SD of triplicate determinations. the mean Ϯ SD of triplicate determinations. 2296 SEMAPIMOD-INDUCED INHIBITION OF c-Raf ACTIVITY

experimental system for identifying the molecular mechanism underlying semapimod-dependent inhibition of proinﬂammatory cytokine production. In vivo concentrations of 1 and 5 ␮M have been demonstrated in preclinical and clinical studies, respectively (30, 31). Therefore, we conclude that 0.1 and 1 ␮M semapimod concentrations used for our in vitro studies have clinical relevance. Semapimod inhibits MAPK signaling pathways in macrophages As it has been previously shown that semapimod blocks p38 MAPK and JNK phosphorylation in vitro (14), this prompted us to study the effects of semapimod on MAPK signaling pathways in more detail. Therefore, the activation status of various kinases involved in MAPK signaling was analyzed by immunoblotting semapimod-treated macrophages using phosphospeciﬁc Abs against MAPK signal transduction molecules. LPS enhances phosphorylation of JNK, ERK, and p38 MAPKs and semapimod treatment resulted in suppressed phosphorylation of all MAPKs (i.e., JNK, ERK, and p38) (Fig. 4). LPS-induced phosphorylation of

upstream MAPK activators was observed (i.e., MEK1/2 for ERK, Downloaded from MKK4 for JNK, and MKK3/6 for p38 MAPK). Impaired phosphorylation of MAPK kinases (i.e., MEK1/2, MKK4, and MKK3/6) was seen upon pretreatment with semapimod. c-Raf phosphorylation was observed in stimulated and control cells (no LPS), in concordance with a previous report (32). Semapimod did

not affect c-Raf phosphorylation, nor did it affect phosphorylation http://www.jimmunol.org/ of PAK, an upstream c-Raf activator (33). These in vitro data suggest that semapimod interferes with MAPK activation upstream from MAPK kinase and downstream from PAK, thereby making c-Raf a likely candidate target. c-Raf is a molecular target of semapimod The observed inhibition of MEK phosphorylation by semapimod in LPS-stimulated macrophages without an apparent accompany- ing effect on c-Raf activation itself may indicate that semapimod is by guest on September 24, 2021 a direct inhibitor of c-Raf catalytic activity. To directly test this hypothesis, we used two protein in vitro kinase assays in which the capacity of recombinant constitutively active Raf to phosphorylate MEK was tested in the presence or absence of semapimod. Incu- bation of active c-Raf or b-Raf together with MEK in the absence FIGURE 4. Semapimod inhibits MAPK phosphorylation in macro- of semapimod clearly induced MEK phosphorylation. Pretreat- phages. The effect of semapimod on LPS-induced MAPK signaling cas- ment of c-Raf with 1 ␮M semapimod for 5 and 10 min abolished cades was studied in macrophages. Cells were pretreated with semapimod ␮ its potential to phosphorylate MEK in this two-protein assay (Fig. (1 and 0.1 M) for 1 h and subsequently stimulated with LPS for 15 min. 5). Importantly, semapimod treatment of b-Raf, an enzyme that is MAPK signaling molecules were analyzed for their activation status on Western blot using phosphospecific Abs. Semapimod suppresses LPS-in- closely related to c-Raf, did not result in altered MEK phosphor- duced phosphorylation of JNK, ERK, and p38 MAPK and the MAPK ylation (Fig. 5), demonstrating the specificity of semapimod as an kinases (i.e., MEK1/2, MKK4, and MKK3/6). Hence, neither the phos- inhibitor of c-Raf enzymatic activity. These data reveal specific phorylation status of c-Raf itself, nor phosphorylation of PAK (an upstream and direct inhibition of c-Raf enzymatic activity by semapimod. c-Raf activator) was affected by semapimod. Blots were reprobed with appropriate Abs to test for equal loading. Western blots represent three Semapimod inhibits c-Raf activity in vivo independent experiments, and duplo conditions are shown. In an earlier study, patients with severe CD (mean CD Activity Index (CDAI) of 380 points) received either 8 or 25 mg/m2 semapimod i.v. once daily for 12 consecutive days (14). Paired did not respond to semapimod treatment. The observed clinical colon biopsies were available at baseline and after 4 wk of treat- response rate correlated to a decrease in C-reactive protein (CRP) ment for six CD patients. Their mean age was 32 years, two were serum concentrations: all responders (five patients) demonstrated male, five were treated with infliximab (anti-TNF), two were decreased CRP levels and the single patient that did not show a treated with steroids, and one was treated with mesalazine before decrease of the serum CRP did not respond clinically (Fig. 6). semapimod treatment. Three patients received 8 mg/m2 semapi- To establish the effect of semapimod treatment on c-Raf activity mod, and the remaining three were treated with a 25 mg/m2 dose. in vivo, colon biopsies were analyzed for phospho-MEK expres- Clinical response was defined by a CDAI reduction of Ն25% and sion. Neutrophils and monocytes were detected as CD14ϩ cells Ն70 points compared with baseline or the occurrence of a clinical (Fig. 7E) and macrophages as CD68ϩ cells (Fig. 7F) in adjoining remission as assessed by a reduction of CDAI of Ͻ150 points (34). sections. Lymphocytes (identified as CD3ϩ cells) had a different Clinical response was observed in five of six patients (mean CDAI distribution pattern compared with phospho-MEK-positive cells reduction of 261 points at week 16), of whom four went into clin- (data not shown). Immunohistochemical analysis revealed high ical remission at 16 wk after initiation (Fig. 6). One of six patients levels of phospho-MEK at baseline, which was mainly localized to The Journal of Immunology 2297 Downloaded from http://www.jimmunol.org/

FIGURE 5. Semapimod-induced suppression of c-Raf kinase activity. In vitro kinase assays were performed to study the effect of semapimod on c-Raf and b-Raf enzymatic activities. Constitutively active Raf (c-Raf and b-Raf) and MEK as a substrate were used to analyze phospho-MEK1/2

Ն by guest on September 24, 2021 (pMEK) expression on Western blot. c-Raf and b-Raf were both capable in FIGURE 6. Clinical responses (as defined by CDAI reduction of 25% Ն phosphorylating MEK in this two-protein assay. Suppressed phospho- and 70 points compared with baseline) were seen in five of six patients. Ͻ MEK (pMEK) expression was demonstrated when c-Raf was incubated for Four of five responders went into clinical remission (i.e., CDAI 150 5 and 10 min with semapimod (1 ␮M) before the in vitro kinase assay. points) at 16 wk after initiation. One responder, who did not go into clinical Semapimod pretreatment of b-Raf did not affect phospho-MEK expression. remission, demonstrated a 205 point decrease of CDAI at week 8 and a 117 Positive (active-Raf with MEK) and negative controls (active-Raf without point decrease at week 16 compared with baseline. The observed response MEK) are shown. Western blots were reprobed with anti-b-Raf and anti- rates correlated to decreased CRP serum levels: all responders demon- c-Raf Abs as a loading control. Similar results were obtained in three strated decreased CRP concentrations during follow-up, in contrast to the independent experiments. nonresponder. As indicated, semapimod was administered for 12 days. macrophages and neuroendocrine cells in the crypts (Fig. 7, A and CD patients, seemed to have significant clinical benefit and that C). Faint phospho-MEK staining was seen in the neutrophil and clinical responses correlated with an inhibitory effect on the p38 monocyte compartment. The decrease in phospho-MEK expres- MAPK and JNK signaling cascades (14). Despite this therapeuti- sion after therapy, observed in five of six patients, was statistically cally relevant outcome, the molecular mechanism of semapimod significant ( p ϭ 0.0348) (Fig. 7G). One patient did not show de- action remains unexplained. We here report that c-Raf in macro- creased phospho-MEK expression (Fig. 7D). Subsequently, we an- phages is the molecular target of semapimod: our studies with alyzed whether the reduction of phospho-MEK-positive cells cor- LPS-stimulated macrophages show that this molecule inhibits LPS related with clinical outcome (defined by CDAI and CRP levels). signaling at the level of c-Raf, resulting in reduced proinflamma- Interestingly, the nonresponder did not demonstrate decreased tory cytokine production. Furthermore, semapimod pretreatment phospho-MEK expression in colon biopsies obtained at week 4 blocked MEK phosphorylation (a Raf substrate) by inhibiting c- after treatment (Fig. 7D) compared with baseline (Fig. 7C). In Raf in a two-protein in vitro kinase assay, whereas the enzymatic contrast, all responders revealed significant reduced numbers of activity of b-Raf (which is structurally closely related to c-Raf) phospho-MEK-positive cells after therapy (Fig. 7H). These data was not influenced by semapimod. Thus, semapimod is a highly indicate that semapimod inhibits c-Raf activity not only in vitro specific c-Raf inhibitor. but also in vivo. In agreement with a role for semapimod as an in vivo inhibitor of c-Raf, colon biopsies obtained from semapimod-treated CD pa- Discussion tients who responded to therapy showed significant decreased As a consequence of the limited efficacy and significant toxicity of phospho-MEK expression, which was predominantly localized to current therapy, there is widespread interest in the development of macrophages and neuroendocrine cells. Interestingly, whereas novel drugs for the clinical management of CD. We have reported semapimod is highly active in the macrophage compartment, our that semapimod, in a small and uncontrolled clinical trial in severe in vitro data confirm an earlier report that T cells are not direct 2298 SEMAPIMOD-INDUCED INHIBITION OF c-Raf ACTIVITY

FIGURE 7. Semapimod treatment significantly decreases phospho- MEK expression in vivo. Paired colon biopsies were obtained at screening (day 1) and at week 4 for six CD patients who received 12 days of i.v. infusions with semapimod. A, High phospho-MEK expression levels were seen before treatment, and this Downloaded from increase was mainly localized to macrophages and neuroendocrine cells (magnification, ϫ200). Inflam- matory cells were identified as neutrophils or monocytes (E) (CD14ϩ, closed arrows) and macrophages (F) ϩ

(CD68 , dotted arrows), in adjoining http://www.jimmunol.org/ sections (magnification, ϫ400). B, Decreased cell numbers staining positive for phospho-MEK were seen in five of six patients following treatment. In contrast, no difference in phospho-MEK expression was seen in one patient before (C) and after (D) semapimod therapy (magnification, ϫ200). G, The decrease in phospho-MEK-positive cells after by guest on September 24, 2021 treatment, observed in five respond- ,ء) ers, was statistically significant p ϭ 0.0348). H, One nonresponder did not demonstrate a reduction of phospho-MEK-positive cells after treatment.

target cells of semapimod action (21). Semapimod treatment did indicate that the cell-specific effects of semapimod are related to not affect cytokine production nor did it affect MAPK signaling Raf isotype specificity and hypothesize that c-Raf inhibition in cascades in T lymphocytes. A likely explanation may be found in macrophages is the primary effector of semapimod action in CD. the relative importance of b-Raf in comparison to c-Raf in acti- Macrophages play a major role in initiating, amplifying, and vating MAPK cascades in lymphocytes, further emphasizing the perpetuating the inflammatory response by activating immune specificity of the c-Raf inhibitory effect observed (35–37). We also cells, including monocytes and T cells (38, 39). However, we are evaluated whether semapimod could affect IL-12 cytokine produc- not aware of data indicating that a therapeutic strategy that mainly tion in activated mature DC, a major pathogenic mechanism in targets macrophages has therapeutic efficacy in a chronic inflam- CD4ϩ lymphocyte-mediated pathology, such as CD (23–26). matory disease in humans. Our current data strongly suggest that Semapimod did not interfere with IL-12 cytokine production, sug- semapimod-induced inhibition of c-Raf in one particular immune gesting that this compound does not influence Th1-mediated re- cell (macrophage) results in a clinical response in severe CD, in- sponses by mature DC in vitro. Taken together, these observations dependent from an effect on T cells or DCs. Hence, the present The Journal of Immunology 2299 study provides novel evidence for a pivotal role of macrophages in 11. Kumar, S., J. Boehm, and J. C. Lee. 2003. p38 MAP kinases: key signalling the pathogenesis of CD (40–45). The identification of c-Raf as the molecules as therapeutic targets for inflammatory diseases. Nat. Rev. Drug Dis- cov. 2: 717–726. molecular target of semapimod raises questions regarding the 12. Manning, A. M., and R. J. Davis. 2003. Targeting JNK for therapeutic benefit: function of this molecule in the inflammatory process. Previous from junk to gold? Nat. Rev. Drug Discov. 2: 554–565. 13. Saklatvala, J. 2004. The p38 MAP kinase pathway as a therapeutic target in work has demonstrated that c-Raf is involved in inflammatory inflammatory disease. Curr. Opin. Pharmacol. 4: 372–377. mechanisms by controlling downstream signaling molecules such 14. Hommes, D., B. van den Blink, T. Plasse, J. Bartelsman, C. Xu, B. Macpherson, as the proinflammatory transcription factor NF-␬B (46–48), G. Tytgat, M. Peppelenbosch, and S. Van Deventer. 2002. Inhibition of stress- activated MAP kinases induces clinical improvement in moderate to severe thereby mediating cytokine synthesis and other proinflammatory Crohn’s disease. Gastroenterology 122: 7–14. mediators (49–55). In addition, various studies have identified c- 15. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen Raf as an important antiapoptotic molecule and its inhibition may by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis well cause effector macrophages to undergo programmed cell factor ␣. J. Exp. Med. 179: 1109–1118. death in the proapoptotic inflammatory environment present in the 16. Berger, T. G., B. Feuerstein, E. Strasser, U. Hirsch, D. Schreiner, G. Schuler, and gut of CD patients (56, 57). As a result, induced apoptosis of B. Schuler-Thurner. 2002. Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories. J. Immunol. Methods 268: macrophages could lead to an attenuation of the inflammatory pro- 131–140. cess. Further studies investigating apoptosis in the gut of semapi- 17. Romani, N., D. Reider, M. Heuer, S. Ebner, E. Kampgen, B. Eibl, D. Niederwieser, and G. Schuler. 1996. Generation of mature dendritic cells from mod-treated CD patients may provide answers to this important human blood: an improved method with special regard to clinical applicability. question. Our data indicate that the proinflammatory effects of c- J. Immunol. Methods 196: 137–151. Raf include not only activation of ERK, but also JNK and p38, and 18. Peppelenbosch, M. P., M. DeSmedt, T. ten Hove, S. J. van Deventer, and J. Grooten. 1999. Lipopolysaccharide regulates macrophage fluid phase pinocy- suggest that c-Raf may be an important target for anti- tosis via CD14-dependent and CD14-independent pathways. Blood 93: Downloaded from inflammatory small molecules. 4011–4018. Clinical studies with semapimod demonstrated that the drug is 19. DeSmedt, M., P. Rottiers, H. Dooms, W. Fiers, and J. Grooten. 1998. Macro- phages induce cellular immunity by activating Th1 cell responses and suppress- relatively well tolerated (14, 30, 58). Side effects included local ing Th2 cell responses. J. Immunol. 160: 5300–5308. irritation at the infusion site (phlebitis) and mild increases in liver 20. Lane, P., C. Burdet, F. McConnell, A. Lanzavecchia, and E. Padovan. 1995. enzymes, both resolving spontaneously within weeks. Preliminary CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation analysis of a large controlled study with semapimod in moderate to and B cell proliferation. Eur. J. Immunol. 25: 1788–1793. http://www.jimmunol.org/ severe CD did not detect clinical benefit. This result is probably 21. Bjork, L., K. J. Tracey, P. Ulrich, M. Bianchi, P. S. Cohen, K. Akerlund, T. E. Fehniger, U. Andersson, and J. Andersson. 1997. Targeted suppression of largely due to the short exposure period (3–5 days), which was cytokine production in monocytes but not in T lymphocytes by a tetravalent allowed in this study design (59). guanylhydrazone (CNI-1493). J. Infect. Dis. 176: 1303–1312. Various Raf inhibitors have passed phase I/II as anticancer strat- 22. Zinser, E., N. Turza, and A. Steinkasserer. 2004. CNI-1493 mediated suppression of dendritic cell activation in vitro and in vivo. Immunobiology 209: 89–97. egy showing a tolerable safety profile (60–68). To our knowledge, 23. Banchereau, J., F. Briere, C. Caux, J. Davoust, S. Lebecque, Y. J. Liu, no clinical studies have been performed with Raf inhibitors as B. Pulendran, and K. Palucka. 2000. Immunobiology of dendritic cells. Annu. anti-inflammatory agents. A principal role for c-Raf in inflamma- Rev. Immunol. 18: 767–811. 24. Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O’Garra, and tory mechanisms in the pathogenesis of CD has important clinical K. M. Murphy. 1993. Development of Th1 CD4ϩ T cells through IL-12 produced consequences, as these data indicate that semapimod and possibly by Listeria-induced macrophages. Science 260: 547–549. by guest on September 24, 2021 other c-Raf inhibitors constitute novel candidates for severe CD, 25. Manetti, R., P. Parronchi, M. G. Giudizi, M. P. Piccinni, E. Maggi, G. Trinchieri, and S. Romagnani. 1993. Natural killer cell stimulatory factor (interleukin 12 and presumably other inflammatory disorders. (IL-12)) induces T helper type 1 (Th1)-specific immune responses and inhibits the development of IL-4-producing Th cells. J. Exp. Med. 177: 1199–1204. 26. Trinchieri, G. 1993. Interleukin-12 and its role in the generation of TH1 cells. Acknowledgments Immunol. Today 14: 335–338. We thank M. Scheffer and J. 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