Lars Petter Jordheim
email: jordheim@yahoo.com

Adriana Plesa

Michel Dreano

Emeline Cros-Perrial

Céline Keime

Stéphanie Herveau

Delphine Demangel

Julie A Vendrell

Charles Dumontet

Sensitivity and gene expression profile of fresh human acute myeloid leukemia cells exposed ex vivo to AS602868

Keywords: Acute myeloid leukemia, AS602868, gene expression, microarray, sensitivity

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy that requires treatment with intensive chemotherapy for cure. While the majority of patients with AML achieve a complete remission with induction therapy, greater than half of these subsequently relapse and ultimately die of the disease [START_REF] Tallman | Drug therapy for acute myeloid leukemia[END_REF]. Relapse is thought to occur because of the failure of chemotherapy to eradicate leukemia stem cells. Current therapies consist in induction chemotherapy, usually a cytarabine/anthracycline combination, followed by consolidation therapy which is required to prevent relapse. Other commonly used compounds include etoposide, anthracycline analogues or mitoxantrone. There are few novel agents in the treatment of adult AML, and only a small subset of patients can be offered curative therapies and for most patients, particularly for the elderly who do not have access to intensive consolidation therapies or allogeneic stem cell grafting, current chemotherapies mainly result in short lasting remissions.

A large majority of elderly patients die of their disease.

The nuclear factor κB (NF-κB) transcription factor pathway has been shown to be implicated in the control of diverse cellular processes, including transformation, induction of proliferation, suppression of apoptosis, cell invasion and angiogenesis [START_REF] Hayden | Signaling to NF-kappaB[END_REF]. NF-κB confers significant survival potential in a variety of tumors, including AML [START_REF] Guzman | Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells[END_REF][START_REF] Cilloni | Nuclear factor kB as a target for new drug development in myeloid malignancies[END_REF]. Accumulating evidence strikingly supports that the NF-κB pathway which regulates cell survival also suppresses the apoptotic potential of chemotherapeutic agents and contributes to drug resistance [START_REF] Nakanishi | Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs[END_REF]. Therefore, direct or indirect inhibition of NF-κB is likely to have antiproliferative activity on cancer cells, such as AML blasts. IκB molecules are repressors of NF-κB transcription factors, and their phosphorylation by IκB kinase 2 (IKK2, IKBKB, IKKβ) induces their ubiquitination and subsequently their proteasomal degradation resulting in NF-κB translocation [START_REF] Schmid | IkappaB kinase beta (IKKbeta/IKK2/IKBKB)--a key molecule in signaling to the transcription factor NF-kappaB[END_REF]. Conversely, inhibition of IKK2 maintains the inhibition of NF-κB by IκB. This has led to the development of IKK2 inhibitors [START_REF] Schmid | IkappaB kinase beta (IKKbeta/IKK2/IKBKB)--a key molecule in signaling to the transcription factor NF-kappaB[END_REF][START_REF] Karin | The IKK NF-kappa B system: a treasure trove for drug development[END_REF]. AS602868 is an anilino-pyrimidine derivative and adenosine triphosphate competitor with an in vitro 50% inhibitory concentration (IC50) of 60 nM toward purified constitutively active IKK2 (IKK2ee). AS602868 displays no or limited inhibitory effects on IKK1 or on a large panel of recombinant kinases. It blocks the phosphorylation of IκB as well as subsequent NF-κB activation in various cell lines and in cells from patients with acute myeloid leukemia [START_REF] Frelin | AS602868, a pharmacological inhibitor of IKK2, reveals the apoptotic potential of TNF-alpha in Jurkat leukemic cells[END_REF][START_REF] Frelin | Targeting NF-kappaB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells[END_REF]. Finally, AS602868 inhibits cell cycle progression and induced apoptosis in multiple myeloma cell lines, confirming its therapeutic interest as monotherapy [START_REF] Romagnoli | Canonical nuclear factor kappaB pathway inhibition blocks myeloma cell growth and induces apoptosis in strong synergy with TRAIL[END_REF][START_REF] Jourdan | Targeting NF-kappaB pathway with an IKK2 inhibitor induces inhibition of multiple myeloma cell growth[END_REF].

To analyze whether NF-κB inhibition could potentiate the effect of chemotherapy, preliminary pharmacological experiments have been performed with AS602868 in combination with the topoisomerase 1 inhibitor irinotecan or its active metabolite SN-38 both in vitro and in vivo [START_REF] Lagadec | Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells[END_REF]. In HT-29 xenografts in nude mice, AS602868 had no significant effect on tumor growth by itself, while it was able to enhance the antitumor effect of irinotecan in a dosedependent manner. Co-treatment of AS602868 with irinotecan allowed a 3-fold reduction of irinotecan dose without a significant loss in antitumor activity. Ex vivo tumor analyses as well as in vitro studies on HT-29 cells showed that AS602868 impaired irinotecan-induced NF-κB activation, enhanced cell cycle arrest and apoptosis, reduced tumor cell proliferation and tumor vascularisation. Preliminary results showed that AS602868 is able to potentiate the effect of etoposide but also of aracytin and doxorubicin on AML blasts as well as the effect of etoposide or vincristine in cutaneous T-cell lymphoma [START_REF] Frelin | Targeting NF-kappaB activation via pharmacologic inhibition of IKK2-induced apoptosis of human acute myeloid leukemia cells[END_REF][START_REF] Sors | Inhibition of IkappaB kinase subunit 2 in cutaneous T-cell lymphoma down-regulates nuclear factor-kappaB constitutive activation, induces cell death, and potentiates the apoptotic response to antineoplastic chemotherapeutic agents[END_REF]. Additional interest for AS602868 in the treatment of AML is related to its inhibition of Flt3 [START_REF] Griessinger | Preclinical targeting of NF-kappaB and FLT3 pathways in AML cells[END_REF][START_REF] Griessinger | AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells[END_REF]. Gene expression profiling is a high throughput screening method used to identify multiple gene expression in a given sample. The comparison of expression profiles between samples can then be correlated with a given biological phenomenon. This approach has proven to be interesting to define molecular subgroups of patients at diagnosis ("expression signatures"), to define the effect of a bioactive molecule (biomarker approach) or to define prognostic or predictive profiles. In the context of determining biomarkers of activity and/or toxicity, pangenomic screening is a potentially interesting approach.

In order to increase the knowledge on the activity and the mechanism of action of the IKK2 inhibitor AS602868, we performed gene expression profiling experiences on clinical samples exposed to this compound in vitro. The present study was conducted to provide data regarding the effect of AS602868 on the expression profiles in fresh human AML cells, and to correlate the baseline expression profile of fresh AML cells with their sensitivity to AS602868 in vitro.

MATERIAL AND METHODS

Preparation of leukemic blasts

Biological samples were obtained from 61 patients with AML at diagnosis (n=32) before initiation of therapy or at relapse (n=29), all followed in the Hematology Department of the Hospices Civils de Lyon, France. Patient characteristics are shown in Supplemental Table 1.

Approval was obtained from Lyon Protocol Review Board, and written informed consent was provided according to the Declaration of Helsinki. Mononuclear cells including leukemic cells were isolated by Ficoll-Hypaque sedimentation from peripheral blood (n=56) and bone marrow (n=5). The median percentage of blast cells in the preparations was 76% (range: 5-99%).

Depending on the arrival of samples, they were assigned to four different groups (A-D). Fifty-one samples were used for in vitro sensitivity studies to AS602868 alone (groups B, C and D), 12 for sensitivity to combinations in vitro (group B), 10 for IκBα determination by flow cytometry (group A) and 20 for gene expression profile studies (group D).

Determination of in vitro sensitivity to AS602868

Fresh leukemic blasts were incubated in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and different concentrations of studied drugs alone or in combinations for 18 hours. Cells were then stained with annexin V / propidium iodide (Roche) and analysed by flow activated cell sorting (FACS) on a Becton Dickinson FacsCalibur ® . Apoptotic cells were defined by annexin V positive cells, irrespective of their propidium iodide status. Results are expressed as the increase of the apoptotic cell population (annexin V positive) substracted for control values (unexposed cells incubated under the same conditions).

Flow cytometry analysis

Total blood from AML samples was incubated with DMSO or AS602868 100 µM for 2 hours at 37 °C, and thereafter for another 15 minutes in absence or in presence of a combination of PMA (1 µM) and ionomycin (500 ng/ml). The incubation with PMA and ionomycin for 15 minutes has previously been shown to induce phosphorylation of IKK [START_REF] Shambharkar | Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways[END_REF]. Samples were diluted in Lyse/Fix Buffer (BD Phosflow) and stored at -80 °C. Flow cytometry analysis was performed for the determination of IκBα expression using IκB-α mouse monoclonal antibody (L35A5, 1:400 dilution, Cell Signaling Technology, Danvers, MA) and Alexa Fluor 488 F(ab')2 fragment of goat anti-mouse IgG (H+L, 1:2000 dilution, Cell Signaling Technology, Danvers, MA) as secondary antibody. Briefly, cells were thawed, then permeabilized using ice cold 90% methanol for 30 minutes on ice, washed and successively stained by incubating cells 1 hour at room temperature for intracellular IκB-α and secondly 30 minutes in the dark for IgG-Alexa488.

Finally, the cells were stained with the surface antibodies (CD45 PerCPcy5.5, clone 2D1, BD; CD3 PE, clone Sk7, BD; CD19 APC, clone SJ25C1, BD; CD4 PE, clone 13B8.2, Beckman Coulter; CD8 APC, clone Sk1, BD; CD34 APC, clone 8G12 anti HPCA2, BD) during 15 minutes at room temperature. Four colour analyses were performed using a CD45/SSC gating strategy and multiparametric analysis of CD3, CD4, CD8, CD19, CD34 and CD45 low blasts cells as well as IκB-α staining. For acquisition and analysis of data we used a Becton Dickinson FacsCalibur ® and CellQuest Pro software.

Toxicity on hematopoietic precursors

The effect of AS602868 on colony-forming cells (CFCs) and long-term culture initiating cells from human hematopoietic precursors was described previously [START_REF] Jeanpierre | BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling[END_REF]. Viability was determined by trypan blue exclusion count and proliferation by Uptiblue assay.

In vitro exposure to AS602868 for gene expression profile study Fresh leukemic blasts (6.10 6 ) were exposed to DMSO-solubilised AS602868 (10 µM) or DMSO (identical volume) in RPMI 1640 supplemented with 10% FCS for 6 hours at 37 °C in 5% CO 2 atmosphere.

mRNA extraction

After incubation with AS602868, cells were placed in lysis solution for RNA extraction.

Total RNA from leukemic cells was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations and subsequently quantified on a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RNA integrity was verified using the BioAnalyser 2100 ™ (Agilent Technologies, Palo Alto, CA, USA).

Pangenomic array experiments

One-color labeled cRNAs were generated from 200 ng of total RNA using the Low RNA Input Amplification Kit (Agilent Technologies) according to the instructions of the manufacturer.

Labeled cRNA were hybridized overnight to Whole Human Genome 4 x 44K microarrays. After washing, microarrays were scanned using the Agilent model G2505B microarray scanner, and data extracted by Feature Extraction software, version 9.5. The default settings were used to scan the microarrays.

Normalization and analysis of microarray data

The quantile normalization method was used to normalize microarray data [START_REF] Bolstad | A comparison of normalization methods for high density oligonucleotide array data based on variance and bias[END_REF]. Two comparisons were performed for the analysis of differentially expressed genes between samples.

First, samples exposed to AS602868 in vitro were compared to the identical samples exposed to DMSO only (paired samples) using a modified Students t-test that uses an empirical Bayesian approach to improve error estimates for individual genes by using information from other genes [START_REF] Smyth | Linear models and empirical bayes methods for assessing differential expression in microarray experiments[END_REF]. Secondly, samples considered to be "sensitive" to AS602868 in vitro were compared to those considered to be "insensitive" samples (unpaired samples). For each comparison a list of genes was chosen when the significance value was less than 0.01. In the next step, the biological relevance of these differentially expressed genes was analysed using Gene Ontology [START_REF] Ashburner | Gene ontology: tool for the unification of biology. The Gene Ontology Consortium[END_REF].

Microarray data are available on Gene Expression Omnibus (GSE1985).

RESULTS

In vitro sensitivity to AS602868

Fifty-one samples were evaluable for in vitro sensitivity to various concentrations of AS602868 and AraC (Supplemental Table 2). All samples were assayed for sensitivity to 30 µM AS602868 inducing a mean percentage of annexin V positive cells of 24.65, with a median value of 26.42 (range: -6.95-71.58). These values are comparable to those obtained with 20 samples exposed to 100 µM AraC (mean: 27.70, median: 21.49, range: -0.33-74.29).

For 9 of the 19 samples incubated with several concentrations of AS602868, we observed a dose-effect correlation with R²>0.6 (range 0.62-0.99). For 8 samples, no correlation was observed (R²<0.6), whereas for one sample we observed a negative correlation (R²=0.92). Among the 20 samples analysed in group D, we observed a positive correlation for 6 samples (range of R²: 0.75-0.98), negative correlation for one sample (R²=0.64) and no correlation for the last 13 samples. For the samples of groups B and C, we did not observe any correlation between sensitivity to AS602868 and to AraC (R²<0.6).

For the 20 samples of group D, we observed a good correlation between sensitivity to 100 AraC and 10 µM of AS602868 (R²=0.74), 3 µM AS602868 (R²=0.76) and 1 µM AS602868 (R²=0.75) but not with 30 or 0.1 µM AS602868. The sensitivity at a concentration of 10 µM AS602868 in this group was used to distinguish between "sensitive" and "insensitive" samples for the expression profile study. The cutoff in terms of annexin V increase was 25%. Using the same cutoff for AraC-induced annexin V, we obtained the same groups of "sensitive" and "insensitive" samples (Figure 1). Using other concentrations for the determination of "sensitive" and "insensitive" samples only slightly modified the groups (data not shown).

In vitro sensitivity in combination with other drugs

Samples of fresh AML blasts from 12 patients were used to study the in vitro sensitivity to AS602868 in association with AraC, etoposide and daunorubicin. Two series of experiments using different concentrations of AS602868 were performed (Supplemental table 3). Correlations were observed between the sensitivity to 1 or 30 µM AS602868 and 1 or 10 µM etoposide and between 1 and 30 µM AS602868 (R²>0.6, range: 0.6082-0.9465). In the first series (6 patients), where three concentrations of AS602868 were evaluated in association with one concentration of AraC, etoposide or daunorubicin, we observed higher cytotoxicity of combinations than for one of the molecules alone only for the combination of 10 µM daunorubicin with 0.3, 3 or 30 µM AS602868 for two patients (Figure 2A, B andC). In the second series (6 patients), where two concentrations of AS602868 were evaluated in association with two concentrations of AraC, etoposide or daunorubicin, we observed increased annexin V staining with AraC for two patients, and with etoposide and daunorubicin for two patients (Figure 2D, E andF).

In vitro toxicity on hematopoietic precursors

Mononuclear cells from normal human bone marrow were exposed to different concentrations of AS602868, and viability and proliferation were evaluated (Table 1). The viability was not modified whereas the IC50 values for proliferation were 5 ± 0.89 and 2.5 ± 0.44 µM at 4 and 7 days respectively. Low concentrations of AS602868 (0.1-1 µM) stimulated the colony formation of total and GM mononuclear cells, but had no effect on CD34 positive cells or the long-term culture initiating cells. At higher concentrations (5-50 µM), these parameters were modified.

Measurement of IκBα in human lymphocytes and blast cells

When active, IKK2 induces the phosphorylation and the degradation of IκBα. IKK2 can be stimulated by PMA and ionomycin and intracellular content of IκBα can be detected by flow cytometry analysis. We developed a method to quantify phosphorylated and unphosphorylated IκBα in lymphocytes and blasts from AML patients and used this to study samples from 10 patients (Figure 3). In lymphocytes, we observed decrease of IκB content by fluorescence after incubation with PMA/ionomycin in all samples. This decrease, reflecting the degradation of IκBα after phosphorylation by IKK2, was partially inhibited in samples co-incubated with AS602868 indicating IKK2-inhibition by this compound. However, in blasts from the same patients, IκBα degradation induced by PMA was only observed in samples from five patients after incubation with PMA/ionomycin. This degradation was reverted by co-incubation with AS602868. In addition, modestly increased signal for IκBα was observed after incubation of lymphocytes from the other patients with AS602868, indicating a higher baseline level of IKK2 activity in these cells than in the other patients or in the lymphocytes from the corresponding patients.

Microarray results

A total of 40 samples corresponding to the cells of the 20 patients exposed to DMSO or 10 µM AS602868 in vitro, were processed for RNA extraction and pangenomic analysis. In a first analysis, we compared samples that were sensitive and insensitive to AS602868 in vitro as identified in the sensitivity assays (Figure 1 and Supplemental Table 2). Two-hundred and eighty genes were found to be differentially expressed between the samples from these two groups (p<0.01 and log2 fold-change >1 or <-1, Supplemental Figure 1A, Supplemental table 4). Log2 fold-changes varied between -4.2 and 3.5. Gene ontology analysis of the 280 genes was performed for major "Cellular components", "Biological processes" and "Molecular functions". This analysis shows that genes differently expressed in sensitive versus insensitive samples show significant enrichment in genes coding for membrane proteins (intrinsic, integral, lipid raft), immune response and response to external stimulus/wounding, organismal physiological processes, receptor and signal transduction activity, including G-protein coupled receptor activity (Supplemental tables 5, 6 and 7).

In a second analysis, we compared gene expression in samples exposed to DMSO alone or AS602868 in vitro in order to determine which genes were regulated by exposure to this agent. Three hundred and fifty four genes were found to be differentially expressed after exposure to 10 µM AS602868 in vitro (Supplemental Figure 1B and Supplemental table 8). Here, log2 fold-changes varied between -2.93 and 2.61. Gene ontology analysis of the 354 genes was performed as above, and this analysis shows that genes differently expressed in AS602868 treated versus untreated samples show significant enrichment in genes coding for extracellular space, immune response, G-protein coupled receptors, regulation of physiological and organismal processes, programmed cell death, response to external stimuli, stress or wounding, protein and RNA binding (Supplemental tables 9, 10 and 11).

DISCUSSION

AS602868 displayed significant apoptotic-inducing activity on fresh human myeloid leukemic blasts, with median activity comparable to that observed with the same concentrations of AraC, a reference compound in AML. In the 51 samples incubated with 30 µM AS602868, the mean increase of the annexin V positive fraction was 26.02%. However, a great variability was observed as individual samples showed from -6.95 to 71.58% positive cells at this concentration (Suppelemental Table 2). Insofar as the clinical use of a compound depends on its therapeutic index, we also evaluated the toxicity of AS602868 on hematopoietic precursors. Inhibition of 50% of the cell proliferation was obtained with 5 and 2.5 µM AS602868. Taken into account the differences in methodology between the study on hematopoietic precursors and fresh lymphoblasts, we find these results rather promising as for a favourable therapeutic index of AS602868. This should, however, be confirmed by extended in vivo studies. In addition, given the properties of NF-κB, its inhibition might have important consequences in vivo. However, initial reports on other compounds inducing NF-κB inhibition indicate good tolerance at least in mice and dogs [START_REF] Guzman | An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells[END_REF].

As the treatment of AML is based on a combination of several drugs, we assessed the sensitivity of blast cells to AS602868 in combination with AraC, etoposide and daunorubicin, all three used in the induction treatment of AML patients. Preliminary experiments on incubations of blast cells with different combinations of these molecules, suggest a greater efficacy of some of these combinations in subsets of patients (Figure 2). Analyses of larger series are required to identify parameters correlated with enhanced sensitivity to these combinations.

Evaluation of IκBα levels by flow cytometry is feasible in AML samples after in vitro

incubation with various compounds and conservation by freezing. We evaluated IκBα levels in lymphocytes and blasts incubated with AS602868. Baseline IκBα levels are relatively similar in lymphocytes from different patients (mean fluorescence intensity (MFI): 44-117) and reproducibly altered by PMA/ionomycin stimulation (range of modified IκBα level: -82.8--41.1%, Figure 3). Lymphocytes can therefore be used as internal controls for studies of modulation in haematological malignancies. IκBα levels vary significantly in blast cells of different AML patients, both in terms of baseline levels (MFI: 35-157), sensitivity to PMA/ionomycin (range of modified IκBα level: -76.5-91.4%) and sensitivity to AS602868. These are imporant characteristics (in addition to selectivity and low variability) for this parameter to be used as a biomarker in a clinical trial of AS602868 in AML patients. Unfortunately, we did not have the cytotoxicity values for AS602868 in the fresh samples used for the biomarker assay, and could therefore not establish correlations between variations in IκBα levels and sensitivity to AS602868.

In the study on differentially expressed genes between sensitive and insensitive samples and between baseline and post-treatment samples, we used a total of 20 fresh adult AML samples assigned as sensitive or insensitive. While studying the difference in expression profile for the two groups of 10 samples, we observed a more than 3-fold higher expression in insensitive cells than in sensitive cells of 11 genes, and a more than 3-fold higher expression in sensitive cells than in insensitive cells for 2 genes (Supplemental table 4). GO analysis of the differentially expressed genes showed significant differences in membrane associated genes (intrinsic or integral, lipid raft, G-coupled receptor activity, signal transducing activity), immune response and response to external stimulus or wounding. Taken together, the following points can be underlined: (i) There is no clear correlation between in vitro sensitivity and altered expression of components of the IKK/NFκb pathway in this series of samples, since these genes or related genes are not found in the list of differentially expressed genes. When looking at differential expression of other kinases, LERK-7, CCRK and PRKCB1 are found to be expressed 2-fold more in insensitive cells than in sensitive cells while LTK is expressed at a higher level in sensitive cells. (ii) Several chemokine ligand genes (CXCL1, CXCL7) or chemokine receptor genes (CCR3, CCRL2) were found to be differentially expressed. (iii) Several solute carrier genes were expressed at higher levels in insensitive cells (SLC35A2, SLC25A37, SLC35E1 and SLC26A8). (iv) Other solute carrier genes (SLC44A5 and SLC5A9) were more highly expressed in sensitive samples. (v) CD52, the target of alemtuzumab, was found to be expressed at a higher level in insensitive cells. If AS602868 was to be tested in lymphoproliferative diseases such as chronic lymphocytic leukaemia, this observation should be confirmed in this setting. While AS602868 was developed as an IKK inhibitor, it also potently inhibits other kinases, such as Aurora A or Flt3 with IC50 values of 5.1 µM and 2.4 µM respectively (data not shown). This might suggest that the activity of AS602868 on tumor cells is due to several mechanisms that should be validated. In particular, mutations in these kinases could modify the cytotoxic activity of AS602868. We only obtained the Flt3 status for 13 samples and could not correlate this with sensitivity. In addition, the inhibition of Flt3 by AS602868 has been shown to be similar for all forms of Flt3 and therefore Flt3 status should not interfere with the activity of AS602868 [START_REF] Griessinger | AS602868, a dual inhibitor of IKK2 and FLT3 to target AML cells[END_REF].

Limitations of this approach include: a) the fact that samples were arbitrarily considered as "sensitive" or "insensitive" to AS602868 on the basis of a given concentration; b) the choice of annexin V induction to evaluate in vitro cytotoxicity; c) the number of samples analysed. The choice of a 10 µM concentration is supported by the fact that this concentration has previously been shown to be the median cytotoxic concentration in this assay. These preliminary data could be confirmed on larger series, by real-time reverse transcription PCR on candidate genes, or by siRNA approaches to explore mechanistic links. In the context of a clinical trial these genes could be compared to the response observed in patients and help determine a profile correlated with sensitivity in the clinic. We used the same 20 samples to study expression profiles after incubation with DMSO (vehicle) or 10 µM AS602868. A total of 354 genes were found to be differentially expressed, with a greater than 1.5-fold increase for 10 genes and a greater than 2-fold decrease for 7 genes (Supplemental table 8). Gene ontology analysis of the differentially expressed genes showed significant differences in extracellular space, immune response and response to external stimuli, G-protein coupled receptors, protein and RNA binding. For this part of our study, we highlight the following: (i) There is no clear correlation between exposure to AS602868 and expression levels of components of the IKK/NFK pathway in this series of samples. These genes or related genes were not found in the list of differentially expressed genes, with the exception of inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IκBKE), for which the expression was reduced 1.26-fold. When looking at differential expression of other kinases, PAI-2 was down-regulated after exposure to AS602868 while SNF1LK, PDPK1, RIOK3, JOSD1, MAP2K1IP1, PANK3, ADRBK1, CLK3, PRKA, AKAP8L and CLK1 were overexpressed.

Variations in the level of expression of these kinases were moderate, since they were less than 2fold. (ii) Several chemokine ligand genes (CXCL10, CCL3, CCL3L3, CCL2, CCL4, CXCL1 and CCL8) or chemokine receptor genes (CCR) were found to be decreased by AS602868 while CKLF was moderately induced. (iii) The solute carrier SLC2A6 was inhibited by AS602868 while ZNT-1, GABA and SLC3A1 were induced by AS602868. (iv) Some serpin-related genes were found to be differentially expressed after exposure to AS602868, including PAI-2, SERPINB2 and SERPINC1. Among heat-shock proteins, two Hsp40 homologues, DNAJB6 and DNAJB14, were found to be moderately induced by exposure to AS602868. Additional preclinical data using siRNA could help to validate the role of certain kinases or other target genes in the cytotoxicity of AS602868 against AML cells. Such a study would be of great value since it could contribute to the identification and validation of novel targets in this disease, and therefore contribute to the development of novel compounds.

As for the first analysis, limitations of this study include the fact that a single concentration (10 µM) and a single exposure time were explored, and that the number of samples analysed was relatively low. On the basis of these preliminary data it would be worthwhile to analyze candidate genes at different time points and using a range of concentrations by real-time reverse transcription PCR (rt-PCR), either in model leukemic cell lines or in fresh leukemic samples. Of note, the strong variation in CYP450 B1 could be related to the metabolism of AS602868.

A striking finding in this work is the influence of AS602868 on the expression of immune related genes and the influence of immune related genes on in vitro sensitivity to AS602868.

Although established in the context of pathological myeloid cells, rather than on normal lymphoid cells, this observation raises the possibility that AS602868 may possess immunomodulatory properties.

Figure 1 .

 1 Figure 1. Distribution of sensitivity to AraC and AS602868 for samples in group D. Isolated

Figure 2 .

 2 Figure 2. Sensitivity of fresh AML blasts to AS602868 alone and in combination with AraC

Figure 3 .

 3 Figure 3. Mean fluorescence intensity (MFI) of IκBα-staining in lymphocytes (A) and blasts

Table 1 . Effect of AS602868 on hematopoietic precursors.

 1 Data are presented as total number of colonies per field and are the mean ± SD of 3 different experiments (3 different marrow samples). DMSO alone was included as control. MNC: mononuclear derived colony forming cell; CD34: CD34 derived colony forming cells; LTC-IC: long-

	term culture initiating cells; nd: not determined
	AS602868	MNC	MNC	CD34	CD34	CD34
							LTC-IC
	µM	total	GM	total	erythroid	GM
	0	9 ± 1.5	9 ± 1.7	74 ± 10	9 ± 1.3	65 ± 7.4 4.6 ± 0.9
	0.1	5.5 ± 1.7 6.5 ± 1.6 66 ± 9.3	8.3 ± 2.0	57 ± 6.3 0.9 ± 0.2
	0.5	23 ± 3.7 22 ± 4.3	nd	nd	nd	nd
	1	19 ± 2.9 20 ± 3.9 61 ± 11	7.7 ± 1.6	53 ± 4.7 7.1 ± 1.3
	5	4.5 ± 0.9 4.5 ± 1.0 55 ± 8.4	5.5 ± 0.9	50 ± 7.1	0 ± 0.3
	10	nd	nd	3 ± 0.8	1 ± 0.2	3 ± 1.0	-
	50	0 ± 0.6	1 ± 0.6	nd	nd	nd	nd

ACKNOWLEDGEMENTS LPJ received grant from Medeco's Stiftelse. This study was supported by research funding from Merck-Serono International S.A. company to Charles Dumontet. The authors acknowledge Dr Véronique Maguer-Satta for studies on hematopoietic precursors. CONFLICTS OF INTEREST Michel Dreano is an employee of the Merck-Serono International S.A. company, whose IKK2 inhibitor is used in this study. Charles Dumontet received research funding from Serono.


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The tradition of future

When considering types of concepts of the future in the Christian tradition, it is useful to distinguish between concepts of the natural world (1.1) and concepts about the realm of history (1.2). Regarding the natural world, we can identify four basic concepts about the end of the world: the renewal or restoration of the world (1.1.1), the destruction or annihilation of the world (1.1.2), the doctrine of the signs of the end (1.1.3), and avoiding discourse about the future of the world altogether (1.1.4).

The tradition of the future of the world 1.Renewal

The renewal or restoration of the world is the most common understanding of the future in Christian history shared by radically different branches of the tradition. It was popularized by Irenaeus of Lyon 1 in his fight against the Gnostic movement and its valuation of the natural world as bad and God-forsaken or the product of an incompetent Demiurge. To avoid these conclusions it was necessary to affirm that the world is a good product of a good creator. If truthfulness is a decisive characteristic of God and nothing in the world in its spatial and temporal extension is independent of God, then God will not annihilate this world, because such an annihilation would mean a self-contradiction in this perfect, good, truthful and coherent divine being.

Since the Reformation, many branches of Christian tradition have adhered to this concept of the future, but the ideas have become less concrete and vivid.

Martin Kähler, writing at the end of the 19 th century, spoke only of 'dramatical alterations in nature' 2 without offering any specifcs. The desire has been to retain the theological aspects of this idea without confessing a specific type of physical worldview.

A contemporary example is provided by the interpretation of the final anthropical principle given by Frank J. Tipler, which can be seen as a secularized variety on this theme of imaging the future. 3

Annihilation

In the last section we saw that the idea of the annihilation of the world was found primarily in the Gnostic movement, so it is somewhat surprising that it reemerged in the Lutheran Orthodox theology of Johann Gerhard (d. 1636).

According to Gerhard the consummation or annihilation of the world is an act of God, by which he transforms heaven, earth, sea and all creatures therein except human beings and angels into nothing by 1 Cf. Irenaeus, adv. haer. V, 20,1. 2 Martin Kähler, Die Wissenschaft der christlichen Lehre von dem evangelischen Grundartikel aus im Abrisse dargestellt, 3rd edn (Waltrop: Spenner, 1994), p. 422. 3 Cf. Frank J. Tipler, Die Physik der Unsterblichkeit: Moderne Kosmologie, Gott und die Auferstehung der Toten, 2nd edn (München: Piper, 2002). the means of fire in order to manifest his truth, his potency and justice and in order to liberate the faithful men. 4 -Gerhard expouses annihilation for the same reason the tradition speaks about a renewal of the world: the truthfulness of God. In Mk 13:31 it is written, 'Heaven and earth shall pass away: but my words shall not pass away.' If this passage is a literal revelation of God's intentions and if God is truthful, then the doctrine of annihilation is the logical outcome of the world. The non-personal world is seen strictly as the means of the well-being of persons here and now, not as necessary for their being. According to Konrad Stock, God's self will become the environment of human and angelic persons, so that there is no further need for something like a world. 5 -To substitute God for the world has implications for the notions of space and time and the notion of the body. If bodies necessarily belong to human beings and if the world is unnecessary in the end for the essence of humanity, then space and time as structures of the world cannot ultimately define the concept of bodies. The conceptual drive for such a new view of space and time is provided in the Lutheran doctrine of Holy Communion and the presence of Christ. Traditionally, the concept of the bodies was defined in spatio-temporal terms, but now it is the other way around: Space and time become secondary deductions from the primary concept of the body or, to be more precise, the concept of the body of Christ. 6

The idea of the (15) signs of the end

In the everyday piety of the Middle Ages, one finds the signs of the end (usually 15), derived from a combination of apocalyptic passages in Scripture such as Mk 13, Lk 21, Mt 24-25, 1 Thess 4:13-18; 1 Thess 5:1-11 and Revelations. These follow the logic of extrapolation from personal experience, especially experiences of life-threatening disasters. As a whole, these signs taken together do not create a coherent picture: events such as rising sea levels, sinking sea levels, the exodus of marine animals from the oceans, bloody dew on plants, earthquakes, crashing rocks and buildings, the madness and stupidity of humanity, graves opening, falling stars, etc. While Thomas Aquinas mentions such signs, he does not assign them the value of a doctrine. He does not expect them to come true and, in fact, considers attempts to calculate the end of the world heretical. 7

The Renunciation of Christian Future-Talk about the World

Since the onset of the modern era, the number of vivid images of the natural future has decreased. So it is not surprising that in the history of theology some theologians have argued Christians should avoid discourse about the future of the natural world altogether, claiming it does not come under the purview of religion.

Such views can be found in the 19th century in the theologies of F.D.E Schleiermacher and Albrecht Ritschl and more recently Ulrich Barth. 8 There is a particula veri to this approach: the logic of extrapolation and the logic of theology do not belong together. Extrapolation must be oriented towards present experience and follows a presumed general principle or law. This approach implies the future is generally accessible to human reasoning. Also, if the presumed general law on which extrapolation is based can be disclosed by non-human entities-as in the apocalyptic movement during the Second Temple period-then there is an implicit contradiction. In the praxis of the Christian faith, revelation does not mean a fixed datable event, but constantly depends on the disclosing activity of God to human beings who are simul iustus et peccator. From this point of view, the logic of 7 Cf. Ludwig Ott, Eschatologie in der Scholastik, (Freiburg u.a.: Herder, 1990), pp. 107-111. 8 Cf. Ulrich Barth, 'Abschied von der Kosmologie -Befreiung der Religion zu sich selbst', in Wilhelm Gräb, ed., Urknall oder Schöpfung, (Gütersloh: Gütersloher Verlagshaus, 1995), 14-42. extrapolation behind the idea of the signs of the end seems not only non-religious, but also irreligious.

Of course, not all attempts to conceive of the future of the natural world follow this form of logic, and I believe we will find systematic arguments that counter this radical view that all theological future-talk must be avoided.

Ideas about the future development of history

Whereas vivid material cataloguing the future of the natural world has decreased since the beginning of the modern era, vivid ideas of the future of history and society seem to be increasing, perhaps even at an inflationary rate.

Certainly, apocalyptic and millennaristic ideas can be found in all periods, though interestingly more often as the exception than the rule. A quite prominent example in this respect is Joachim of Fiore.9

In considering the historical record, there are two matters to consider: First, there was no exceptional millenaristic movement at the end of the first millennium.

Secondly, the picture changes if we differentiate carefully between apocalypticism, denoting the view that one can gain methodologically sound knowledge about the laws governing the course of history based on some form of epistemic reason, and parousial imminentism, which denotes the conviction that one is living in the end times of the second coming of Christ: Whereas parousial imminentism has existed from the beginnings of Christianity and through the Reformation, apocalypticism has been increasing in an inflationary manner since the onset of modernity. It has even become a doctrine with multiple, distinctive forms such as premillenarianism (i.e. a pessimistic view in which vice increases throughout the course of history so that the second coming of Christ is necessary to bring about a final, positive time in the new millennium), postmillenarianism (i.e. an optimistic view in which virtue increases throughout the course of history so that the second coming of Christ happens after the historically achieved millennium), dispensationalism (i.e. the Assuming my distinction between apocalypticism and parousial imminentism is correct, however, we should follow the opposite thesis instead: Why not view the rise of Christian apocalyptic forms in the modern era as "baptized" manifestations of truly secular-modern theories? In my opinion, this thesis is supported by the fact that modernity can also be described as an attempt to attain the faculty of reasoning and apply it to all areas of human experience, including the realm of future history. One might explain the simultaneous decrease of vivid ideas of the future of the natural world in theology and philosophy with the simultaneous rise of the sciences. Similar to the epistemic approach to the signs of the end, the logic behind Christian and secular apocalypticism follows the logic of extrapolating from present experience according to a proposed general principle or law.

Correspondingly, my critique of all forms of apocalypticism is the same as described above.

Ontological commitments and interests

No one can examine historical facts except through the lens of their own presuppositions, ontological commitments and interests. So I am going to make some of my presuppositions explicit in order to evaluate the tradition further and find an appropriate approach to Christian discourse about the future. The concept of action can be seen as a basic anthropological notion of humanity's being and living in the world. Human action can be described in many ways. As there is no way to reduce this concept naturally, concepts such as intention and personhood are necessary. 12 I do not want to point out the traditional problems discussed in the philosophy of mind. Rather, I would like to stress a single point which is necessary though not sufficient to describe human action: All human action happens within a specific horizon of anticipation and expectation.

The dimension of the future in the concept of human action

All action reflects future intentions. Because these intentions are only necessary conditions, in addition to other circumstances, for the event which will happen in future, the horizon of expectation of future is a necessary element for the concept of human action. 13 Since we can expect things in the future with an attitude of hope or fear, this horizon of expectation is divided into two kinds of affective human approaches. For reasons that do not need to be explored here, Christian theology can call this horizon of expectation a horizon of hope but not fear.

Since theology can be defined as the self-explication of Christian practiceunderstood as the sum of all being in the world for Christians-there is no theology without concepts of the future. If this presumption is correct, eschatology can be described as a specific perspective on Christian practice as a whole, rather than a particular set of doctrines. Eschatology deals with the whole of Christian practice from the perspective of expectation just as dogmatics deals with the whole of Christian practice from the perspective of faith (which can be seen as another necessary precondition of the concept of action) and just as religious studies deals with the whole of Christian practice from the perspective of Christian practice as cooperation and interaction interwoven with different religious worldviews.

Consequently, not talking about the future-including the future of the natural realm-is not an option at all. Or better put: the aforementioned rejection of discourse about the future of the world as a theological topic should not be seen as 12 Cf. Edmund Runggaldier, Was sind Handlungen? Eine philosophische Auseinandersetzung mit dem Naturalismus (Stuttgart u.a.: Kohlhammer, 1996), pp. 42-105.122-145. 13 Cf. Mühling, Eschatologie, pp. 35-40. a true rejection but as nothing more than a modulation of the theme of annihilation or destruction. Whereas Christian discourse about annihilation or destruction as a mode of future-talk is problematic because it sees the natural world as ontologically irrelevant for the realm of religion, the rejection of all future-talk is problematic because it sees the natural world as epistemically irrelevant for the realm of religion.

Both options appear to belong to the same set, in fact, if there is no categorical difference between epistemology and ontology, because all epistemology presupposes ontological commitments and all ontology needs epistemological reflections.

While Christian theology cannot abandon the theme of the eschatology of the natural world, neither can it base eschatology on the logic of extrapolation.

Eschatical 14 horizons of expectation

In order to solve the problem of how theology can talk about the future of the world, we must differentiate further the concept of the horizon of expectation. We have already mentioned that this horizon of expectation is structured based on the affective distinction between fearing and hoping. Human affectivity shapes this horizon of expectation, but so does human reason. It was been suggested

distinguishing between four modes of expectation, derived from observations of everyday language: 15 1. The unsurprising: there are some things we simply expect with more of less certainty: that a lecture will end, for example, or that Tuesday will follow Monday, etc. We have certain sure evidence in our horizon of expectations that such events will happen. They are located close to the center of our horizon of expectations.

2. The prospectively surprising: Some events may be very improbable, but we are able to imagine them. It is very improbable that Barack Obama will appear in the publishing house of this journal and that he-out of gratitude for the new 14 In regard to the material hope 'eschatical' is used on order to denote the object-language whereas 'eschatological' is used in regard to the theological analysis of the eschatical state of affairs. 15 Cf. Mühling, Eschatologie, pp. 41-48. insights gained by this journal-will appoint one of its contributors as his new

Minister of Defence and Philosophy. But, however improbable it may be, the fact that I am able to imagine it signifies that, were it to happen, it would have to be classified as a prospective, imaginable surprise. Events of this kind lie within our horizons of expectations but they are located closer to the margins.

3. The absolutely retrospectively surprising: Some events are absolutely surprising. These can only be events that lie completely beyond our horizon of expectation. I cannot provide an example of such an event. If I could, it would mean it had to be classified under one of the other subsets of events. Therefore, we have to call it 'absolutely retrospective' because we can address such events as surprises only after they have happened. Nevertheless, it seems that expecting the absolutely retrospective surprising is essential human. Poor would be the person who does not want to be surprised by life in this absolute manner. Absolutely retrospectively surprising events broaden our horizons of expectations and are essential elements of our life experience. Viewed from the present, such events can be seen as located outside our horizons of expectations.

4. The relatively retrospectively surprising. There is a fourth kind of future event in our lives which plays a key role in the explanations to follow. It is a combination of the first and the third kind of surprising events. We expect to be surprised by some future events in a retrospective way, because we have sufficient evidence that they will happen, but we do not know in what manner. Good birthday presents belong to this kind of event: I expect a future event belonging to the first type, e.g. my birthday this year in the future. Therefore, I expect to get birthday presents, but I have no idea what those wonderful gifts will be. So they belong to the realm of the relatively retrospectively surprising.

As I described these four kinds of surprising events, I changed from talking Since there are some things within this horizon of expectations (e.g. the Christevent), I expect some events situated outside my ultimate horizon of experience which share different kinds of attributes: some attributes we can talk about because their linguistic logic is based on revelation, and attributes we cannot talk about. The source of the constitution of such ultimate or eschatical horizons of expectations can be seen as disclosure experiences in our lives, belonging to the category of relatively retrospectively surprising events.

Whereas all our particular horizons of expectations can be broadened by future experience, our eschatical horizon of experience cannot be broadened by surprising events in the future. Or, rather, it can be changed by surprising events in the future, but this is not a kind of broadening but a kind of conversion. If we all have such implicit eschatical horizons of experience constituted by relatively retrospectively surprising experiences of disclosure, and if the Christian faith provides its genuine horizon of expectation, and if human life cannot be understood without reference to the natural world, then Christian theology is able to talk about the future of the natural world in a methodologically rigorous manner: All we have to do is seek the common structure of the constitutive disclosure experiences and ask whether these are able to provide conceptualizable descriptions for discourse about the future of the world.

We can expect that the notions uncovered in such an analysis will contain three types of analogies: positive analogies, since talk of them is based on that part of the relatively retrospective surprising which lies within our ultimate horizon of expectation; negative analogies, since we can exclude some attributes as belonging to the kind of events that will surprise us in an absolutely retrospective way; and neutral analogies, i.e., attributes which may or may not belong to the retrospectively surprising part of events we hope for. With its positive, negative and neutral analogies, this kind of logic of Christian future-talk resembles the logic of the models and metaphors explained in the theory of scientific models offered by Mary

Hesse and others, 16 but with two decisive differences. First, whereas the neutral analogies can probably be transformed into positive and negative analogies by future experience in the realm of science, this is not possible for Christian futuretalk. Secondly, there is a fourth type of characteristic of events we hope for in Christian future-talk which we cannot talk about at all, as they belong strictly to the absolutely retrospective.

Revelation and its structure

The search for a common structure of revelation constituting the Christian horizon of expectation has to start with the everyday experience of Christians. All

Christians see their lives within the framework of the story and stories of the gospel narrated by fellow-Christians. Whether or not they are consciously aware of it, with the help of ideas by Robert Jenson and Christoph Schwöbel 17 we can identify a threefold narrative structure of the story of the Gospel: 1. Jesus Christ is seen as the ultimate personal disclosure of the eschatical: he is the eschatos by his implicit claims about being the kingdom of God, both in his person and his actions. 2. This kingdom of God proclaimed by Jesus Christ is the kingdom of the God of Israel, a god who is addressed as his Father, the creator of the world. 3. The result of his claim is his death on the cross, which seems to suggest the failure of his claims. 16 Cf. Mary B. Hesse, Models and Analogies in Science (London u.a.: University of Notre Dame Press, 1963), pp. 9-20. 17 However, Christians claim that he has been resurrected. Asked for the reason for this belief, Christians answer the activity of God itself, which has disclosed that Christ is alive (Gal 1:15f). Since this activity of God among believers has not come to an end but continues to generate faith in living people today, the lives of people today merge with the story told to them, which is also related in this story of the gospel itself. The threefold narrative structure of the gospel can also be valuated as an identity descriptor of the Christian God, abbreviated with the use of proper names, and understood as the story of the Father, the Son and the Holy Spirit.

Furthermore, this threefold structure of the gospel is not simply a narrative, but implies ontological and eschatical claims because revelation suggests that the revealing instance communicates in a truthful manner and that the product of this revelation is the constitution of the eschatical horizon of expectation.

While the ontological claims have to be examined in the doctrine of God, here we are concerned primarily with the ultimate or eschatical implications: beyond that threefold ground of hope, Christians have nothing to hope, fear or expect. The Son, Father and Holy Spirit are the Ultimate. The Son is an eschatos, the Father is an eschatos, and the Holy Spirit is an eschatos. Therefore, they can be named as the three eschatoi. 18 The logical structure between these three eschatoi is not an addition. They are constitutively related to one another through a relationship the Christian tradition describes with different terms. The Eastern tradition speaks about the two relationships of gennesis and ekporeusis, the Western tradition since Richard of St.

Viktor has tended to speak of a single relationship of processio, providing the necessary personal proprieties with the help of the filioque. 19 One could discuss which of these descriptions is more appropriate; one could also discuss whether we have to add other or new descriptions of this eschatical or ultimate relationship.

However, for now I want to focus on the logical structure of these descriptions of this relationship, independent of its semantic content: 18 Cf. Mühling, Eschatologie, pp. 73-74. 19 1. The story of the Father is not the story of Christ or the Holy Spirit and the story of Christ is not the story of the Father or the Holy Spirit. The same holds true for the story of the Holy Spirit. Therefore, the relationship between them is one of asymmetry.

2. The Father is related in a way to the Son and the Spirit in which he is not related to himself. Also, the Son is related in a way to the Father and the Spirit in which he is not related to himself. The same holds true for the Spirit's relationship to the Father and the Son. Hence, this ultimate relationship is a relationship of irreflexiveness. definitions and uses only syntactical ones. For reasons we do not have space to explore here, I believe Christian theology can add two more characteristics to these three formal marks of the ultimate or the eschatical: first, this ultimate relationship can also be described as an open event 20 and second, it probably also implies the characteristic of being partly irreversible.

In sum, God, the divine essence or the ultimate or eschatical reality, can be experienced as a necessary condition of possibility for Christian hope and the Christian horizon of expectation and contains 3+2 formal characteristics: God is an asymmetrical, irreflexive, transitive event-like relationship between Father, Son and Holy Spirit that may also be partly irreversible.

The ultimate future of the world

The eschatical fulfilment of the world in God

A possible answer to the question of the future of the world depends on how we define the relation between time and eternity and the relation between space and omnipresence or infinity, because the natural world can be described in the first instance with the help of spatio-temporal terms. In the history of theology, philosophy and the sciences we find different ways of solving these questions.

Regarding the question between time and eternity there are three main paradigms or models:

1. Eternity can be seen as the negation of time. This concept can be illustrated by Augustine's famous Book IX of his Confessions and it opens the way for some concepts of eternity in the mystical tradition. Cf. Mühling, Gott ist Liebe, pp. 276. 308-315. 21 Cf. Aurelius Augustinus, Confessiones Buch 11, lat.-dt., ed. b. Flasch, Kurt (Frankfurt am Main: Klostermann, 1993 Many theologians follow this pattern, sometimes with modifications: Whereas the 'pure type' conceives of eternity as the sum of time, so that all times are present in God's eternity and there is no room for an open multioptional future, Duns Scotus, Tillich, Pannenberg, process-philosophy, Peter T. Geach and others speak only of a co-presence of the past and future in eternity, although the future is only the realm of all different possibilities known to God.23 3. Most recently, others such as Richard Swinburne have adhered to this model of eternity as an endless progression of events. Swinburne distinguishes between the metric of time identified with the existence of the world and the topology of time, i.e. the succession of events identified with eternity. [START_REF] Cf | The Christian God[END_REF] In the history of the relation between space and omnipresence, we likewise find different kinds of solutions:

1. One can define space in different ways as a finite absolute being or as an infinite absolute being, a definition that appears in various versions in the thought of Newton, Samuel Clark and also in Einstein's general theory of relativity (GTR), though it was contrary to his own intentions. Under these circumstances, God's infinity can be seen as the negation of space or it can be identified with the endlessness of space itself. 25 2. The other possibility is to speak of space as a purely derivative concept of the relations between things in keeping with Leibniz, Mach, Einstein's original intentions, and others. Under these circumstances it is possible to conceive of God's infinity as a set of relations and therefore a 'space' of its own, connected by God's economic action to the space of creation. [START_REF]Briefwechsel mit Leibniz[END_REF] In all these cases, our understanding of God's eternity and infinity largely depends on our understanding of time and space, commonly attained by means of the philosophy of nature. This is precisely the point I wish to criticize: If, according to the eschatological logic of theology, we have to start with revelation, then it must be possible to say something about eternity and infinity in antecedent way.

This does not mean that a philosophy of nature based on scientific theories cannot contribute anything to theological understandings of eternity and infinity, but that these concepts cannot be the basis for theological thinking. In order to follow this path, we have to resist the temptation to ask the ontological question about the essence of space and time that Augustine did, for example ("I want to know the real meaning and the nature of time" [START_REF] Flasch | Was ist Zeit? Augustinus von Hippo, das XI. Buch der Confessiones. Historisch-philosophische Studie[END_REF]. Having reservations about ontological concepts of the philosophy of nature is no obstacle to using philosophical and scientific concepts in theology, but rather a precondition for using such concepts at all. Similarly, the scientist also should remain modest and resist providing answers too quickly about the nature of space and time, which always imply specific ontological baggage.

For a theologian it seems much easier to answer the question of the understanding of eternity and infinity implied in the eschatical horizon of expectation which is constituted by the revelation of the eternal God. If we can meaningfully apply the traditional attribute of God's oneness to the ultimate source of disclosure, and if oneness means the identity of the extensions of all concepts applicable to God, then God's essence must be eternity and infinity. And if the divine essence, in whatever way it may be described semantically, shares the logical structure of an asymmetrical, irreflexive, transitive event-like relationship between Father, Son and Holy Spirit that may also be partly irreversible, then infinity and eternity are also structured in the same way.

In a second step we can look at different spatiotemporal concepts of the philosophy of nature. Without talking about the nature of space and time, we can say that all discourse about space and time presupposes that both share this structure of asymmetry, irreflexiveness and transitivity. In both Einstein's special theory of relativity (STR) and Minkowski's 'world,' this structure is not denied. The question, however, is whether irreversibility also has to be an essential characteristic of time. The STR and GTR may be interpreted as reversible. But if we take into account the second law of thermodynamics, we have evidence that we should consider the observed irreversibility as real. [START_REF]Zeit und Ewigkeit[END_REF] Similar circumstances apply to questions of space in quantum theory: Einstein's point in criticizing quantum theory with the help of arguments along the lines of EPR-paper is not to save a deterministic world against a view of the world essentially structured by chance. His point is to preserve spatial location as the only and ultimate source of individuation of all conceivable objects. [START_REF] Cf | Nicht Sein Kann Was Nicht Sein Darf", Or the Prehistory of EPR 1909-1935: Einstein's Early Worries about Quantum Mechanics of Composite Systems[END_REF] But the implication of the non-separability of objects implied in quantum theory does not restrict space to an asymmetric, irreflexive, transitive, but reversible relation, which is the condition of possibility for individuation. Rather, it allows ontological interpretations, giving events, relations and processes priority over individual objects. In sum: without answering questions about the nature of space and time, we can assert that space and time share the logical structure of irreflexive, asymmetric and transitive relations. In addition, space is unquestionably reversible. It is uncertain whether time is really irreversible, but something can be said for this opinion. If we were to offer an interpretation in light of the theology of creation we could perhaps say the following: God created a world with the structures of space and time that corresponds to the formal structure of God's own being. Since God is not only the first but also the last, meaning the unsurpassable Being which reveals our eschatical horizon of hope, we can assert that our ultimate future is to be incorporated 30 into the logical structure that is God-or the love and life of God-itself. Since both our present life in time and the ultimate, non-extrapolated future share the same logical characteristics, the benefits will be the same: We will be fulfilled persons-in-relation within the framework of the relationship of the ultimate itself. There will be personal transcendence, alterity and communication, there will be also an event-like, processual structure, due to the transformation of judgment without ambiguity.

One question remains: What denotes 'us' and 'we' in the last sentences? Only personal beings? Or also non-personal entities? I do not have an exact answer. But given that our being in space and time is essentially related to natural, non-personal entities, and given God's revelation for us, and given that the biblical tradition also speaks of the intrinsic value of some non-personal beings for God independent of human and personal beings (Ps 104:26), then I assume that the natural world will 30 Christoph Schwöbel objected that, -whereas the language of incorporation is meaningful in the context of ecclesiology, especially in the speech of the church as the body of Christ -, it should be avoided in eschatology. I can imagine two main reasons probably prohibiting this concept in the eschatological context: 1. The eschatical inclusion into the inner-trinitarian framework of love is a passive and gratuitous one. 2. The unity of the inner-trinitarian framework of the divine being is no organic unity of a body. The first objection can be rejected, because also an incorporation can be a gratuious one, as it is the case also in the ecclesiological speech of incorporation. The second objection is a serious one. However, if (1) humans become incorporated into the body of Christ, and if (2) the body of Christ belongs to the human framework of relations of the second person of the trinity (his 'human nature'), and if (3) the human 'nature' belongs to the identity of the son implying, that there is no eschatical logos asarkos, then also human beings become incorporated into the love and live of the trinity. They are already incorporated into the love and live of God in an indirect manner and they will become incorporated in a direct manner. The new state of affairs in the eschatical reality is the shift from directness to indirectness, not from being not-incorporated to being incorporated. As a last remark one has to point also to the fact that all language -consequently also the language of incorporation -is metaphorical language including more or less epiphoric and diaphoric parts. Using language in a complete meaningless way is as impossible as using language in a complete literal way. The meaning is dependent on the context, and the context of theological reasoning consists in conceptual explication by providing theoretical models. also partake in this eschatical transformation into the logical structure of God. We have no idea of the extent, however. The answer to a child's question of whether there is any place in God for his pet can consequently answered with: 'We do not know, but there probably is.'

The syntactical answer as a regulative concept for semantical answers

The syntactical answer to the question about the ultimate, non-extrapolative future of the world was that the world will be transformed and incorporated into the logical structure which is the ultimate reality itself, since the ultimate reality as well as the space and time of the world share the same logical structure. I want to propose that this concept can be used as a regulative concept for both theological concepts and dialogue with scientific concepts of the future of the world.

On the one hand, regarding different ideas of piety, biblical language, the theory of pastoral counseling and theology, we can say that the concept of an ultimate future proposed in this paper opens the door for a number of different metaphors used in different contexts of religious speech: It corresponds to biblical language about the ultimate future such as being with Christ forever in the case of the parousia (1 Thess 4:17), seeing God face-to-face (1 Cor 13:12), the new heaven and the new earth (Isa 65:17; 2 Peter 3:13; Rev 21:1), the eschatical city of Jerusalem (Rev 3:12; 21:2, 10-27), eternal life (Rom 6:22; 1 John 5:20), and the eschatical communication under the vine branches (Mi 4:4; Zech 3:10). It also provides space for some theological concepts of the ultimate future as in the visio beatifica or some views of theosis. 31 Also the concept of being incorporated into (the) [hi]story of the life and love of God, which appeared as a key-concept in some recent theories of pastoral counseling 32 will fulfill the condition of this ultimate fulfilment of the world in God. The extent to which the specific characteristics of these different 31 Cf. Thomas Aquinas, s.th., IIIs q92. There is no unified concept of theosis in the history of Eastern theology, but see, for example, Anastasius vom Sinai, Wegweiser (Viae Dux Adversus Acephalos), PG 89 (Paris: Imprimerie Catholique, 1865), p. 36 who teaches divinization without a personal merging of creator and creature. 32 Cf. Wolfgang Drechsel, Lebensgeschichte und Lebens-Geschichten: Zugänge zur Seelsorge aus biographischer Perspektive (Gütersloh: Kaiser, Gütersloher Verlagshaus, 2002), p. 365. ideas reflect positive, negative or neutral analogies is not the point of this paper. I believe the more vivid the pictures become, the more weight may be placed on the negative analogies. But in any case, there must be a specific amount of very vivid characteristics despite the fact that these are unknown to us in our present condition.

On the other hand, other semantical ideas of the future of the world are necessarily excluded by the proposed syntactical regulative idea: ideas of the soul's becoming one with God described with the analogy of a drop of wine merging into an infinite sea found in the mystical theology of Heinrich Seuse, 33 or ideas such as partaking in God's consequent nature as a moment in God's memory found in process-metaphysics. 34 Furthermore, the idea of no longer having a personal status but gaining something impersonal or vanishing completely also have to be excluded, because they are incompatible with the ultimate event-like structure of asymmetry, irreflexiveness, transitivity and perhaps irreversibility.

Regarding dialogue with scientific concepts of the future of the world, the concept of the eschatical fulfilment of the world in God has to be applied in a less straightforward way. On the one hand, we have to stress the independence of theological future-talk: this eschatical fulfilment of the world is independent of prognostic ideas such as a 'big crunch' and 'heat-death', because the symbols of resurrection and last judgment as symbols of continuity and transformation do not imply a gradual and observable transition of the pre-ultimate, extrapolative future to the ultimate or eschatical future. On the other hand, we have to stress that scientific concepts about the future very quickly become trans-scientific concepts of the philosophy of nature, loaded with manifold and differing ontological and perhaps hidden religious baggage. Obviously, this is the case with ideas such as Tipler's final anthropic principle, as well as in less apparent instances. In this context, I wish to argue for the necessity of a joint inquiry into these different 33

Once again: renewal or destruction?

I began this paper by introducing the two traditional concepts of renewal and annihilation without deciding between them. In light of the proposed concept of the eschatical fulfilment of the world in God, can we move forward? I think the answer is 'yes' and 'no.' The answer is 'yes' because, compared to our syntactical concept, both ideas contain some positive analogies: Johann Gerhard's statement that the world becomes superfluous because God will be the environment of human beings is correct because space and time, whatever it will be, are indeed superfluous to the eschatical fulfilment, except its logical structure. The doctrine of renewal is correct, because there is a shared structure between time and eternity and this may be interpreted as the survival or renewal of the world. Furthermore, personal entities will not partake alone in this fulfilment, but non-personal ones will as well though to an unknown extent. But the answer to our question must also be 'no' because, in light of the concept of the eschatical fulfilment of the world in God proposed here, the decisive differences between the two traditional ideas seem to vanish, so there is no reason to decide between them. Furthermore, compared to the concept of the eschatical fulfilment of the world in God, the ideas of renewal, annihilation and all millenaristic ideas about the history of the world do not belong to the realm of the eschata, the ultimate things, but remain in realm of the preultimate (in contrast to ideas such as the parousia of Christ, the resurrection of the dead, the last judgment and the fulfilment of the kingdom of God). They are preeschata.

  according to different dispensions), etc. 10 Because similarities exist between Christian apocalypticism and some concepts of history by Enlightenment philosophers-most prominently those in the philosophies of history of Lessing, Hegel and their followers as well as pessimistic thinkers such as Spengler and perhaps Huntington-some scholars have called these philosophies secularized forms of Christian apocalyptism. 11

  about one horizon of expectations to talking of horizons of expectations. The reason is that I believe we have different horizons of expectations, depending on our involvement in different communities, different times of action and different relationships to different people. But we can assume that there is something like an implicit all-inclusive horizon of expectations. This all-inclusive horizon of expectations can be called the ultimate or eschatical horizon of expectations. It is constituted by experiences of disclosure, which can be seen as revelation. In the case of the Christian horizon of expectations, all our previous personal experience is connected to the story of the gospel of Christ as the ultimate basis of revelation.For example, since Christ has been resurrected by the Father and the Spirit, I expect to experience something similar to his resurrection although this is completely different from all my previous and present experiences. That example illustrates the constitution of my ultimate or eschatical horizon of expectation.

  Cf. Robert W. Jenson, 'The Triune God' in Robert W. Jenson/Carl E. Braaten, ed., Christian Dogmatics, vol. 1, (Philadelphia: Fortress, 1984), 79-191; Christoph Schwöbel, 'Trinitätslehre als Rahmentheorie des christlichen Glaubens' in Christoph Schwöbel, ed., Gott in Beziehung, (Tübingen: Mohr-Siebeck, 2002), 25-51.

  3. a) Coming to faith means experiencing the activity of the Holy Spirit. But the Gospel communicated in the Christian discourse of God tells us that the Holy Spirit is related to Jesus Christ. Therefore, experiencing the activity of the Holy Spirit always implies experiencing the activity of Jesus Christ. For this reason, the main churches of the Christian tradition had refused to talk about any activity of the Holy Spirit as unconnected to the activity of Christ. b) Furthermore, since the story of Jesus Christ implies the story of the God of Israel as Creator, experiencing the activity of Jesus Christ means experiencing the activity of the God of Israel. For this reason, the church has rejected the view of Marcion and others that there can be Christian faith without reference to the God of Israel. Given a) and b) we can conclude that, since experiencing the activity of the spirit means experiencing the activity of Christ, and since experiencing the activity of Christ means experiencing the activity of the Father, then experiencing the activity of the Spirit means also experiencing the activity of the Father. In other words, the relationship between Father, Son and Spirit is a relationship of transitivity. The logical structure of the eschatoi, beyond which we have nothing to hope or expect, is the structure of an asymmetrical, irreflexive and transitive relation. Admittedly, in comparison to many descriptions of God from Christian tradition such as 'life' or 'love,' this definition does not seem very vivid and illustrative. But the advantage of this very formal description is that it can include many vivid descriptions of God from language derived from the practice of piety and theology, because it avoids all semantic

  21

2.

  Eternity can also be seen as the abundance of time. Main examples of this model are the Boethian conception of time and eternity inspired by Plotinus. 22 20

  -religious commitments and also for a Christian philosophy of nature based on revelation.

4

  Johann Gerhard, Ioannis Gerhardi Loci Theologici cum pro adstruenda veritate tum pro destruenda quarumvis contradicentium falsitate per theses nervose solide et copiose explicati (Lipsiae: Hinrichs, 1885), XXIX, p. 26: 'Consummatio seculi sive destructio mundi est Dei actio, qua per ignem coelum, terram, mare et omnes creaturas, quae in eis sunt, solis angelis et hominibus exceptis, in nihilum rediget ad veritatis, potentiae ac justitiae suae manifestationem et piorum hominum liberationem.'

	F o r
	P
	e
	e r
	R
	e
	v i e
	w

5

Cf. Konrad Stock, Annihilatio Mundi: Johann Gerhards Eschatologie der Welt (München: Kaiser, 1971), p. 123.

6

Cf. Stock, Annihilatio Mundi, pp. 164-167.

  Studienausg. (Düsseldorf u.a.: Artemis & Winkler, 2004), p. 262 and Plotin/Werner Beierwaltes, Über Ewigkeit und Zeit (Enneade III, 7), 4th edn (Frankfurt am Main: Klostermann, 1995), pp. 98-99.

).

22

Cf. Anicius Manlius Severinus Boethius, Consolatio philosophiae -Trost der

Philosophie. lat.-dt.,

  Cf. Heinrich Seuse/Loris Sturlese/Rüdiger Blumrich, Das Buch der Wahrheit. mittelhochdt.-dt. = Daz buechli der warheit (Hamburg: Meiner, 1993), p. 69.

34

Cf. James A. Keller, Problems of Evil and the Power of God (Aldershot u.a.: Ashgate, 2007), pp. 136-167.

submission for International Journal of Systematic Theology

Cf. Joachim von Fiore, Concordia Novi ac Veteris Testamenti, ND Venedig 1519 (Frankfurt/M.: Minerca, 1964), b. 5, p. 84. p. 112.

A short introduction to the theology of apocalypticism is provided in Markus Mühling, Grundinformation Eschatologie: Systematische Theologie aus der Perspektive der Hoffnung (Göttingen:Vandenhoeck & Ruprecht, 2007), pp. 198-220.

Cf. Volker Leppin, Chiliasmus III.3, in RGG, 4th edn, vol. 2, 1999, 139-40.

Cf. Calvin Normore/Norman Kretzman, The Cambridge History of Later Medieval Philosophy (Cambridge: Cambridge University Press, 1982), p. 362; Paul Tillich, Systematische Theologie III, 4th


Robert W Jenson

ONCE MORE THE LOGOS ASARKOS

2 union with Jesus, was an historic mistake, if perhaps an inevitable one. Great genius has subsequently been devoted to the task of conceptually pasting together God the Son/Logos and Jesus the Son/Logos of God, and we may be thankful for many of the ideas posted along the way. But the task itself is wrongly set and finally hopeless.

Two. In whatever way the Son may antecede his conception by Mary, we must not posit the Son's antecedent subsistence in such fashion as to make the Incarnation the addition of the human Jesus to a Son who was himself without him. By the dogma, Mary is the mother of God the Son, she is Theotokos, and not of a man who is united with God the Son, however firmly. Thus the church confesses that God the Son was himself conceived when Mary became pregnant -even if theology often labors to evade this confession's more alarming entailments. That Mary is Theotokos indeed disrupts the linear time line or pseudo-time line on which we westerners automatically -and usually subliminally -locate every event, even the birth of God the Son; but that disruption is all to the theological good.

Three. The sentence "How would the Trinity have been the Trinity if God had not created a world, and there had therefore been no creature Jesus to be the Son, or had let the fallen creation go, with the same result?" is often taken for a real question. And here I do have a retraction to make. In the past I have sometimes responded to the supposed question, saying that God would presumably have somehow been the same triune God that he is, but that we can say nothing further about that "somehow". I now think that even this response concedes too much to our unbaptized notion of time, by supposing that the collection of words quoted at the beginning of this paragraph actually makes a question which one can answer, however sparingly. It has now dawned on me that the putative question is nonsense, and so therefore is my previous attempt to respond to it.

Four. Occam's razor is a liberating and necessary tool. Indeed thou shalt not posit metaphysical entities beyond necessity. The ground of this commandment is theological. In Christian theology the warrants of ontological necessity must finally be scriptural, and we are not allowed to proceed past what they can support. In the present case, belief that there is the Son is indeed mandated by Scripture. But what mandates belief in an intrinsically disembodied metaphysical entity that is "the" Logos? The passage often cited is the Prologue of John. But O* Logos there is God's creative utterance from Genesis 3 1, which John glosses; even if middle Platornism's deity of that name lies somehow behind John's usage, it is the text and not its possible background that is to be read.

So much for my charactistic maxims. But then there is a maxim of a different and even overriding sort, that is also not negotiable: the eternity of the Son/Logos. In whatever sense "was" and "when" obtain within the life of God, there indeed never was when the Son was not; and it is vital to remember that in the "pre-" folk worry about about there is nothing but the life of God. Some critics have seized on my insistence that theTheotokos means what it says, to claim that I am a crypto-Arian; they have forgotten that point.

The problem is how to unite these two sets of non-negotiables. In the traditional language, how are we to conceive the "pre-existence" of the Son who Jesus is? How are we to read the Prologue of John's Gospel and the narrative it introduces as a coherent whole? Or indeed read the Prologue itself as a coherent whole?

In my Systematic Theology 2 I advanced three more or less related proposals. I continue to think that what I wrote on those pages is true. But the proposals are insufficient to one part of their joint task, and my language was intended to show that I suspected as much. I should have explicitly said so. A quick look at each of the proposals follows, differently ordered than in the Systematic Theology.

My exploitation of Romans 1:3-4 is -I dare to suggest -a necessary and significant corrective to the tradition's usual way of thinking about the Son's divinity. These two artful verses -whether they were devised by Paul or found as an existent confessional formula -are perhaps the New Testament's closest approach to a "two-natures" doctrine. The Son is, "according to the flesh," that is, as a creature, constituted as such by human descent, and indeed by specific descent from David. So far so easily read within the standard tradition. But then note what is paired with this! He is "determined " 3 to be "the - ----------2. 1:141-144.

3. Not "declared" as in NRSV, ready as always to blunt Scripture's more offensive statements. Within the mystery of the triune God's specific eternity, the Son's subsistence must therefore be as much from the Spirit as telos as from the Father as arche. Or anyway, so the Scriptures' pervasive eschatological-pneumatological orientation suggests -an orientation scrupulously ignored by traditional doctrine of the Son's divinity. Thus attending to Romans 1:3-4 is in itself a much needed move. The only thing is: the passage is not about the Son's pre-existence, which is what people are worried about.

The second proposal is more directly about pre-existence, and is in my judgment again a needed observation and corrective. For the most obvious pre-existence of Christ attested by Scripture is his active presence in old Israel: as the Glory of the Lord, the Angel of the Lord, and the Word of the Lord. 5 These appearances were recognized by the rabbis as one reality, God's Shekinah, God as his own "dwelling" among his people, as one who is other than God yet is the same God. Patristic exegetes even recognized in the Glory's "appearance" to Ezekiel 6 as "a man" the actual man who would again appear in God's Glory on the Mount of Transfiguration.

Without fully exploiting the fact of Christ's active and identifiable presence in Israel, and the scriptural testimony to the presence, doctrines of his pre-existence will always be biblically rootless. We must not merely note that Christ is present in the Old Testament; we must shape our understanding of his - ----------4. The phrase often translated "with power", as if there was or could have been the divine Son of God without power, is adjectival and should be translated that way. Just as "Spirit of holiness" is in the diction of this confession equivalent to "Holy Spirit". 5. And as other, more structural, phenomena.

6. In the call vision, to which see my commentary on Ezekiel, ad loc. Here is where my third previous proposal fits -and fails. In the Systematic Theology I suggest a "pattern of movement" within the divine life. The problem is not that the suggestion is merely false, but that it is -as has several times been pointed out -hopelessly vague. It either gets us nowhere, or if it does may get us to a wrong place -though insistence that it must mislead is not quite warranted. So what to do? Here goes.

It is not as an individual instance of humanity as such, not as one among many who have the same human nature 7 , that Jesus is the second hypostasis of the Trinity. According to Aquinas -and to me -a divine hypostasis is "a subsisting relation", that is, a relation that is its own term, and so is not an instance of anything at all 8 . If then we obey my opening maxims, particularly the first, we will say that it is Jesus' relation to the Father -and not Jesus as a specimen of humanity -which is the second hypostasis of Trinity. The Father's sending and Jesus' obedience are the second hypostasis in God.

Next step: this relation itself can indeed subsist "before" Mary's conception, in whatever sense of "before" obtains in the Trinity's immanent life. For that life is constituted in nothing but the web of such relations, which as terms we are told to call Father, Son and Spirit. In the divine life there is therefore no line on which the relation describable as God's sending and Jesus' obedience could occupy a position "after" anything. And again we must remember that antecedent to God's life, there is no realm in which the Son/Logos might "pre"-exist, or not. 9 -----------7. Whatever "human nature" is. 8. Except, I suppose, the class of subsisting relations -which is a small class, with three members. 9. Some of my critics seem not to have gotten it altogether into their thinking, that time is created.

Admittedly, it is a hard thing to do. Yet the Father himself does not subsist otherwise than as a relation to the Son -the circle is the very point. Therefore, since there is no way in which anything could be precedent to the Father, there is nothing precedent to the Son as subsistent relation.

I am not sure whether or not this analysis fits what folk have in mind as the "pre-existence of the Logos. But then, I have never been sure what anyone who is not an antecedently convinced neo-Platonist can actually have in mind under that rubric. I continue to regard Logos asarkos , used for something "before" the Incarnation on any sort of line, as a Vorstellung in futile search of a Begriff.

  of God" by action of the Spirit; and so not by divine origin as the christological tradition might make us expect, but rather by the Resurrection, the supreme act of the Spirit who is "Giver of Life".

  to his life there. Still, we do indeed have to account ontologically for this presence. It would appear that a pre-existence of the Old Testament pre-existence is called for.

  triune life has ontological pre-cedence to the Son as subsistent relation, is the "monarchy" of the Father: his relation to Jesus is the condition of the possibility of Jesus' relation to him.