EP1523306A1

EP1523306A1 - Use of modulators of the signal transduction path using the protein kinase fyn for the treatment of tumorous diseases

Info

Publication number: EP1523306A1
Application number: EP03771094A
Authority: EP
Inventors: Martin Eilers; Bernd Berwanger; Holger Christiansen; Oliver Hartmann; Helmut Schäfer
Original assignee: Morphochem AG fuer Kombinatorische Chemie
Current assignee: Morphochem AG fuer Kombinatorische Chemie
Priority date: 2002-07-24
Filing date: 2003-07-24
Publication date: 2005-04-20
Also published as: ZA200050690B; CA2492833A1; DE10233737A1; CN1671366A; US20060084591A1; JP2006500331A; IL166314A0; AU2003258537A1; WO2004010986A1

Abstract

The use of modulators of the signal transduction path using the protein kinase Fyn (such as Rho-kinase inhibitors or inhibitors of the protein kinase Csk, for example) for the treatment of tumorous diseases, in particular for the therapy of neuroblastomas is disclosed.

Description

Use of modulators of the signal transduction pathway via the protein kinase Fyn for the treatment of tumor diseases

The present invention describes the use of modulators of the signal transduction pathway via the protein kinase Fyn (such as, for example, Rho-Kinase inhibitors) for the treatment of tumor diseases, in particular for the therapy of neuroblastomas.

Neuroblastomas are malignant cancers of the peripheral sympathetic nervous system that occur in childhood. They are one of the most common childhood malignancies. Nationwide, approximately 150-200 children develop this tumor annually, which is largely incurable in advanced stages. The course of the disease varies depending on the individual case and ranges from spontaneous regression to progressive course and the formation of metastases. The tumor develops from progenitor cells of the autonomous nervous system, which controls the involuntary functions such as cardiovascular, bowel and bladder activity. A total of approximately 40% of the children affected fall ill within the first five years.

A genetic criterion that serves as a reason for an unfavorable prognosis is the amplification of the MYCN

Gene that leads to the deregulated expression of the N-Myc protein in the tumor tissue. N-myc is a transcription factor that can control gene expression both positively and negatively. Using a cell culture model, it was shown that Myc proteins regulate both cell growth and proliferation.

Current clinical investigations use three criteria for a prognosis of the neuroblastoma: the stage of the tumor, the age of the patient and the amplification of the MYCN gene. However, the amplification of the MYCN gene is not a reliable criterion, since tumors can also develop that do not have an amplified MYCN gene.

In contrast, the object of the present invention is to use genetic differences in those signal transduction pathways which control the proliferation and differentiation of the neuroblastoma for the prognosis and therapy of tumor diseases, in particular of the neuroblastoma.

The object of the present invention is to provide new methods for the treatment of tumor diseases, in particular of the neuroblastoma.

Microarray analysis is used to measure the parallel expression of a large number of genes in any number of samples from many patients, all of whom have neuroblastoma. This is followed by a statistical analysis in which clinical parameters are correlated with the expression data and thus genes are identified that are causally involved in the course of the tumor. By identifying the genes that are causally related to the course of the tumor are involved, we open up the possibility of a causal therapy of the disease.

The expression of the genes found correlates with specific tumor stages. Surprisingly, the majority of these genes belong to a signal transduction pathway that runs via the protein kinase Fyn. This signal transduction pathway is known to regulate cell adhesion, cell proliferation and differentiation in cell cultures. Changes in cell adhesion are responsible for the formation of metastases, cell differentiation and proliferation control the actual tumor growth. It is shown that the data can be validated by independent measurement methods. Western blot methods were used to show that there is a connection between the activity or expression of the Fyn kinase and the stage of the tumor. All measurements show that signal transduction by Fyn is switched off in advanced tumor stages. The expression analyzes underline the role of the Fyn signal transduction pathway in the formation of metastases.

In addition, we demonstrate that Fyn has a causal role in the above-mentioned processes in neuroblastoma. Using two different cell lines from the neuroblastoma, it is shown that the expression of Fyn, ie the reactivation of the signal transduction leads to a growth arrest, to increased adhesion and to the differentiation of neuroblastoma cells in culture. We have thus proven that the loss of signal transduction by Fyn is the cause of the biological processes mentioned. Using a microarray analysis of human tumor samples from the neuroblastoma, it was surprisingly found that the signal transduction pathway regulates tumor growth and the formation of metastases via the protein kinase Fyn. It was also found that signal transduction by Fyn is switched off in the advanced tumor stage. Influencing the signal transduction path via Fyn Kinase, in particular the (re-) activation of the signal transduction path regulated down in the neuroblastoma via Fyn, is a therapeutic option for the treatment of neuroblastomas.

By using modulators of the signal transduction pathway via the protein kinase Fyn, it is possible according to the invention to prevent tumor growth and the formation of metastases, and thus to treat tumor diseases (in particular neuroblastoma).

A further embodiment of the invention relates to modulators of the signal transduction path via the protein kinase Fyn, the modulators being inhibitors of enzymes which are inhibited directly or indirectly by active Fyn.

Another embodiment of the invention relates to modulators which lead to an increase in Fyn activity in the tumor cells.

Another embodiment of the invention relates to modulators, the modulators being inhibitors of protein kinase CSK, inhibitors of Rho-Kinase, inhibitors of MAP-Phosphatase or activators of Protein Kinase C are.

Another embodiment of the invention relates to modulators which inhibit the proteolytic breakdown of Fyn kinase in vivo.

A further embodiment of the invention relates to modulators, the modulators being inhibitors of phosphatases which counteract Fyn.

Another embodiment of the invention relates to modulators which contribute to an increase in signal transduction by Fyn.

In addition, the invention relates to the use of modulators of the signal transduction pathway via protein kinase Fyn for the production of a medicament for suppressing the metastasis of tumors in the early stages, the tumor being a pediatric or an adult tumor.

The invention further relates to the use of modulators as described above as a component of a pharmaceutical composition, characterized in that the composition in addition to the modulator as an active ingredient optionally comprises carriers and / or adjuvants.

Another embodiment of the invention relates to modulators as described above, characterized in that the modulator in the form of a pharmacologically acceptable salt, Solvates, hydrates, or a pharmacologically acceptable formulation.

A further embodiment of the invention relates to modulators as described above, characterized in that the modulator is present as a prodrug, comprising the modulator and at least one pharmacologically acceptable protective group which is split off under physiological conditions.

A further embodiment of the invention relates to modulators as described above, characterized in that the modulator is administered orally, parenterally, rectally, by inhalation, transdermally or intranasally.

Detailed description of the invention

Corresponding scientific work on this topic has been published by B. Berwanger, 0. Hartmann, E. Bergmann, S. Bernard, D. Nielsen, M. Krause, A. Kartal, D. Flynn, R. Wiedemeyer, M. Schwab, H. Schäfer, H. Christiansen and M. Eilers: "Loss of a FYN-regulated differentiation and growth arrest pathway in advanced stage neuroblastoma" in Cancer Cell, Vol. 2, November 2002, pages 377-386, to which reference is made in full becomes.

In order to gain insight into the molecular sequence of the development of neuroblastomas, expression profiles of 94 individual tumor tissue samples were created using a human, unigenous 4608 cDNA chip. Each chip is hybridized with cDNA as a reference, which differs from one human neuroblastoma cell line (SHEP). The tumors were selected to reflect the distribution of the tumor stages and the MYCN amplification of the entire tumor bank. To allow comparisons between the individual spots and the arrays, each signal was corrected for its background, and the log-transformed on / off regulation intensity ratios were calculated and standardized. AT statistics with two samples and adjusted p-values were used to identify differently expressed genes (Callow et al., 2000). The adjusted p-values simultaneously correct the testing of 4608 genes and estimate the overall probability of detecting a wrong gene (see methods).

We found 123 differently expressed genes when we compared MYCN-amplified (n = 17) and unamplified (n = 77) tumors (adjusted p-value <0.05). These genes were used in an uncontrolled, hierarchical cluster, and analysis showed that all but three of the tumors were correctly assigned to one of the two classes. A functional attribution showed that most of the genes that were switched on in MYCN-amplified tumors code for proteins that play a role in protein synthesis, metabolism and in

Play regulation of the cell cycle; this is consistent with the microarray data that resulted from the use of inducible systems in cell culture. These results show that Myc proteins regulate both cell growth and proliferation, and that these are determined by the Cell culture models developed to extend to the development of the neuroblastoma. The expression of a number of genes which were identified as target genes of (c-) Myc in cell culture experiments was significantly different in both classes of tumors. A large group of genes, most of which were switched off in MYCN-amplified tumors, code for proteins that are involved in multistage signal transduction pathways. This shows that Myc proteins represent a negative feedback signal for signal transduction pathways. Surprisingly, the cell cycle genes that are more abundant in MYCN-amplified tumors encode proteins that are known to act as a checkpoint response, or in the G2 or M phase of the cycle, which indicates a new function of Myc in control and in the late cell cycle.

Deregulation of these genes can easily reflect the advanced tumor stage of most MYCN-amplified tumors (Table 1). To rule out this possibility, we compared their expression between stage 4 of the MYCN-amplified and unamplified tumors. The expression of all genes we analyzed was regulated by MYCN amplification, regardless of the tumor stage. Conversely, deregulation could reflect direct regulation by N-Myc. It is in agreement with this idea that multiple E-boxes are present in the promoter and the introns of the MAD2, CENPE and AURORA2 genes (FIG. 1, field e). In fact, chromatin immunoprecipitation showed that N-Myc linked to the E- Boxing of the MAD2 genes is bound. We have concluded that at least some of the genes we identified are direct target genes of N-Myc.

Table 1

Table 1: Clinical parameters of 94 patients who participated in this study. Age and stage are very confused, and due to the small number, they could not be analyzed independently.

An initial analysis showed that stages 1 and 4, but not stages 2, 3 and 4S, of the non-MYCN-amplified tumors show differences in their expression profiles (data not shown). We found 36 significant genes that were expressed differently in tumors in stage 1 (n = 19) and stage 4 (n = 21) (adjusted p-value <0.2). This set of genes showed little overlap with the group of genes that differentiated MYCN-amplified from unamplified tumors; a functional attribution of the genes showed that the genes coding for proteins that are involved in metabolism and Protein synthesis plays a role, from the group of stage-specific genes were apparently absent, in contrast to the genes that were characteristic of MYCN-amplified tumors. In contrast, a characteristic percentage of genes that were expressed differently codes for genes that are involved in signal transmission by the non-receptor tyrosine kinase Fyn and the actin cytoskeleton; these genes were down-regulated in a coordinated manner in the advanced stage of neuroblastoma. This group includes Fyn itself, the actin filament binding protein (AFAP), a protein that binds to and activates Src and Fyn kinase; α-catenin (CTNNAl), an actin-binding protein whose binding to β- and γ-catenin is regulated by Fyn-dependent phosphorylation; the neural cell adhesion protein NRCAM, which is signaled by non-receptor tyrosine kinases and the actin-binding proteins tropodulin and MARCKS.

The Western blot confirmed the reduced expression of Fyn in stage 4 tumors compared to stage 1; in addition, the experiment consistently found a slower migration of the Fyn protein in extracts from all stage 1 tumors compared to stage 4 tumors, indicating that the autophosphorylated (active) form is present. Phosphatase treatment confirmed that the different migration was due to phosphorylation. In accordance with these observations, high Fyn kinase activity was consistently obtained in tissue samples from stage 1 tumors using immunocomplex kinase assays that used both Au- measure tophosphorylation and phosphorylation of the exogenous substrate, enolase (Wolf et al., 2001). In contrast, the Fyn kinase activity in extracts from stage 4 tumors was variable and, on average, significantly lower.

To test whether Fyn plays a role in the differentiation and regulation of cell proliferation of neuroblastoma cells, we used a transient transfection to express Fyn in SH-SY5Y cells - a human neuroblastoma cell line that is made up of one Stage 4 tumor is derived (Pahlman et al., 1981). The expression of wild-type fyn induced the formation of multiple neurites and obvious morphological characteristics of the differentiation. Staining with antibodies directed against cyclin A as a marker protein of cell proliferation revealed that the cells expressing active Fyn had exited the cell cycle. In contrast, cells expressing a kinase negative allele (FynK299M) showed no signs of morphological differentiation.

Consistent with the role of AFAP in the activation of Fyn, expression of a constitutively active allele of AFAP induced morphological changes that are strongly reminiscent of active Fyn, while a dominant-negative allele of AFAP does not affect differentiation.

We repeated the experiments in IMR-32 cells, a human neuroblastoma cell line that carries an amplified MYCN gene (Clementi et al., 1986). Similar to the results obtained in SH-SY5Y cells, expression of the active ones was induced Fyn-Kinase neurite formation and an exit from the cell cycle. This shows that the induction of differentiation by Fyn also takes place in the presence of an amplified MYCN gene. This agrees with the finding that the expression of FYN, AFAP, NRCAM and CTNNAl is down-regulated equally in MYCN-amplified as well as in non-amplified tumors, with respect to tumors in stage 1.

Overall, we identified two genetic programs that regulate the development of neuroblastomas, one that is controlled by the amplification of the MYCN gene, and a second, stage-specific program of gene expression. Both programs are largely independent of each other because (a) both sets of genes are minor

Overlap shows that (b) the genes are down-regulated by MYCN amplification regardless of tumor stage, and (c) the tumor-stage specific genes are down-regulated regardless of MYCN amplification. The deregulated expression of MYCN activates genes that code for proteins that are involved in both cell cycle progression and growth and suppresses genes that code for proteins that are involved in multi-step signaling processes in a human tumor. These findings specifically show that Myc proteins control gene expression in the G2 phase of the cell cycle and activate genes that are involved in checkpoint processes. Our data show that Fyn kinase regulates the proliferation and differentiation of neuroblastoma cells in vivo; this is also supported by the finding that the stage-specific expression profile predicts survival. Detailed expression profiles of individual tumor stages showed that Fyn down-regulation is most noticeable between stages 1 and 2, which correlates with the formation of metastases in the local lymph nodes. The down-regulation of Fyn as well as the altered cell adhesion control the formation of local metastases in vivo.

Active Fyn can perform its function in several ways: In neuronal cells, non-receptor tyrosine kinases Rho-GAP phosphorylate, which leads to the inactivation of Rho and the induction of differentiation. It has been found that there are molecules in signaling pathways that are inhibited by Fyn signaling, making them candidates for therapeutic intervention. Influencing the signal transmission path downward from Fyn provides, according to the invention, a therapeutic access for advanced neuroblastomas.

Preferred target molecules in the Fyn signal transduction pathway are those which are inhibited by Fyn signal transduction.

Preferred modulators are inhibitors of enzymes which are inhibited directly or indirectly by active Fyn. Further preferred are modulators which lead to an increase in Fyn activity in tumor cells (preferably in neuroblastomas).

Inhibitors of the protein kinase CSK, which is expressed in neuroblastomas and is a negative regulator of Fyn in vivo, are particularly preferred.

Also preferred are modulators that inhibit the proteolytic degradation of Fyn kinase in vivo, such as. B. general inhibitors of the proteasome (LLNL, MG132) or specific inhibitors of the E3 ligases involved.

Further preferred modulators are inhibitors of phosphatases, which counteract Fyn such as. B. MAP Kinase Phosphatase 1.

Again preferred modulators that contribute to an increase in signal transduction by Fyn such. B. Rho kinase inhibitors, MAP phosphatase inhibitors or activators of protein kinase C.

Furthermore, the present invention also includes pharmacologically acceptable salts, solvates, hydrates or pharmacologically acceptable formulations of the modulators described.

Examples of pharmacologically acceptable salts are salts of physiologically acceptable mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid or salts of organic Acids such as methanesulfonic acid, p-toluenesulfonic acid, lactic acid, acetic acid, trifluoroacetic acid, citric acid, succinic acid, fumaric acid, maleic acid and salicylic acid. The modulators according to the invention can be solvated, in particular hydrated. Hydration can occur, for example, during the manufacturing process or as a result of the hygroscopic nature of the initially anhydrous compounds. If the modulators described contain asymmetric carbon atoms, they can be present either as diastereomer mixtures, mixtures of enantiomers or as optically pure compounds.

The pharmaceutical compositions according to the present invention contain at least one of the modulators described as an active ingredient and, optionally, carriers and / or adjuvants.

The pro-drugs, which are also the subject of the present invention, consist of a modulator according to the invention and at least one pharmacologically acceptable protective group which is split off under physiological conditions, e.g. an alkoxy, aralkyloxy, acyl or acyloxy group, e.g. an ethoxy, benzyloxy, acetyl or acetyloxy group.

The present invention also relates to the use of the modulators for the production of medicaments for the prevention and / or treatment of tumor diseases (in particular neuroblastoma). In general, the modulators are made using the known and acceptable modes, administered either individually or in combination with any other therapeutic agent. Such therapeutically useful agents can be administered in one of the following ways: orally, for example as dragées, coated tablets, pills, semi-solids, soft or hard

Capsules, solutions, emulsions or suspensions; parenterally, e.g. as an injectable solution; rectally as suppositories; by inhalation, e.g. as a powder formulation or spray, transdermally or intranasally. For the production of such tablets, pills, semi-solids, coated tablets,

Dragees and hard gelatin capsules, the therapeutically usable product can be mixed with pharmacologically inert, inorganic or organic drug carriers, for example with lactose, sucrose, glucose, gelatin, malt, silica gel, starch or derivatives thereof, talc, stearic acid or its salts, dry skimmed milk and the like. For the production of soft capsules, drug carriers such as vegetable oils, petroleum, animal or synthetic oils, waxes, fats, polyols can be used. For the preparation of liquid solutions and syrups, drug carriers such as water, alcohols, aqueous salt solution, aqueous dextrose, polyols, glycerin, vegetable oils, petroleum, animal or synthetic oils can be used. For suppositories, drug carriers such as vegetable oils, petroleum, animal or synthetic oils, wax, fat and polyols can be used. Compressed gases suitable for this purpose, such as oxygen, nitrogen and carbon dioxide, can be used for aerosol formulations. The pharmaceutically usable agents can also contain additives for preservation, stabilization Containing emulsifiers, sweeteners, flavorings, salts to change the osmotic pressure, buffers, coating additives and antioxidants.

Combinations with other therapeutic agents may include other agents that are commonly used to treat tumor diseases (especially neuroblastoma).

For the prevention and / or treatment of the diseases described above, the dose of the modulators according to the invention can vary within wide limits and can be adjusted to individual requirements. In general, a dose of 0.1 μg to 100 mg / kg body weight per day is suitable, with a preferred dose of 0.5 to 10 mg / kg per day

Day is. In suitable cases, the dose can also be below or above the values given above.

Examples

Examples of Rho Kinase inhibitors are described in EP0370498, US4997834, EP0956865, US6218410, US4678783, US6153608, EP0885888, WO0168607 and WO0156988. Specifically, compounds I (Fasudil), II (Hydroxyfasudil] III and IV are mentioned here:

- 1y

Examples of activators of protein kinase C are compound V, the compound EP-70905 from Europeptides, the natural substances bryostatin, teleocidine, alysiatoxin and esters from phorbol and ingenol. Other compounds such as VI and VII are in J. D. Winkler et al. J. Am. Chem. Soc. 1999, 121, 296-300.

An example of a MAP kinase phosphatase 1 inhibitor is the compound MX 7091 from Maxima Pharmaceuticals.

An example of a CSK inhibitor is the natural product staurosporine.

material and methods

Microarray experiments

The chip used contains the cfNA set gf200 from Research Genetics (http: //www.resgen. Com), as well as 100 additional cDNA, which have already been described as being suitable for a prognosis of neuroblastoma development (for

For details, see http: // www. imt .uni-marburg. de). Each cDNA was applied twice per chip. The chips were as described in Hegde et al. , 2000 described, using a GMS 417 arrayer.

An initial histochemical examination of a series of 100 randomly selected tumors showed that approximately 95% of the tumors contained less than 5% cells in the tissue samples that were not tumor cells (see Bergmann et al., 2001). Therefore, no further attempt was made to dissect the tumor tissue before preparing the RNA. All of the RNA from the neuroblastoma tissue and the SHEP was isolated using a Qiagen RNA isolation kit according to the manufacturer's instructions. 40 µg of total RNA was used to label Cy3 and Cy5 fluorescence Produce cDNA according to the published protocol (http://brownlab.stanford.edu). The chips were scanned using a GMS 418 fluorescence scanner and the images were analyzed using the IMAGENE 3.0 software. The expression data were either by

Northern blot analysis or confirmed by Real Time RT-PCR assays.

Standardization and quality control ImaGene 3.0: The software parameters such as "signal ranges" or "spot detection threshold" (see ImaGene user manual for details) were optimized for maximum reproducibility before the image analysis of our experiment. The average signal and background intensities for both channels are obtained for each spot. To determine the differences between the spots, the corrected background ratio of the two channels was calculated and log ₂ - transformed. In order to equalize the fluorescence intensities of the two dyes and to enable a comparison of the expression level in cross experiments, the raw data were standardized. First, we used a pin-wise (pinwise) intensity-dependent standardization (Yang et al., 2002) to correct an inherent tendency on each chip (the lowess scatter-plot smoother). In a second step, general standardization was carried out in order to center the logarithmic ratios for each array at 0 (in order to take general coloring and scanner effects into account). Since each gene was applied twice on the chip, the mean logarithmic ones Ratios M calculated from the copies. If the copies deviated more than four times or the background intensity was higher than the signal intensity, the gene was excluded from this array.

Statistical analysis

The final data matrix consisted of 4608 standardized gene expression measurements (log ₂ ratios) from 94 individual tumors (with missing values). To compare the expression profile between two independent groups, a t statistic with two samples for each gene was used. To take multiple testing into account, we calculated the adjusted p-values for each gene using a step-down permutation algorithm (Westfall and Young, 1993, Algorithm 4.1). This strategy was previously applied to microarrays (Callow et al., 2000). The permutation algorithm provides strict control of the family-wide error rate (FWER) and takes into account the correlation of the variables (genes). The process does not rely on an assumption of normality; it is assumed that the t-statistics have the same zero distribution asymptotically for all genes (or that the p-values are monotonic in the observed t-statistics about the genes).

Cluster analysis

Before the cluster analysis, the expression profile of each gene was centered by subtracting the mean observed value. The average, linked hierarchical grouping into clusters (average linkage hierarchical clustering) was then carried out for genes and chips, using the Euclidean distance measure as implemented in the J-Express program (Dysvik and Jonassen, 2001).

Western blots, immunoprecipitation, phosphatase treatment The following antibodies were used in western blots, immunofluorescence experiments and immunoprecipitations: α-Fyn: (sc-434); CC-cdk2 (sc-163), CC-cyclinA (sc-751), all from Santa Cruz. Neuroblastoma tissue was lysed as described (Bergmann et al., 2001).

For the phosphatase treatment, 500 μg of cellular proteins were incubated overnight at 4 ° C. with 5 μg of FYN antibody, which is bound to protein G beads. Immune complexes were either incubated with λ protein phosphatase (NEB) or with λ protein phosphatase and phosphatase inhibitors. Immune complexes were separated by means of a 10% SDS PAGE, transferred to a PVDF membrane and detected with an α-Fyn antibody.

In vitro kinase assays

500 ug of cellular proteins were immunoprecipitated with 5 ug α-Fyn antibody bound to protein G beads, washed and equilibrated in kinase assay buffer, incubated for 15 minutes with 10 ug Ci-γ-ATP (Amersham) and 0.125 mg / ml enolase , separated by electrophoresis using a 10% SDS PAGE and dried. The results were visualized on a Fuji Phosphoimager and quantified using image calibration software. Chromatin Iππnunpräzipitation

Chromatin immunoprecipitation was carried out as previously described (Bouchard et al., 2001). Nuclear extracts were immunoprecipitated overnight at 4 ° C with 3 ug a-N-Myc antibody or control antibody bound to Protein A and Protein G beads. For the PCR analysis, a specific primer pair for intron 1 of prothymosin alpha (as a positive control), as well as primer pairs which amplify the specified regions of the MAD2 gene, were used. Primer sequences are available on request.

Cell culture experiments SH-SY5Y and IMR-32 neuroblastoma cell lines are in RPMI

1640 cultivated, which is supplemented with 10% heat-inactivated FCS. CMV-controlled expression constructs which code for Fyn wild type (Fynwt) and FynK299M have been described (Wolf et al., 2001). Plasmids encoding AFAPΔLZ and AFAPΔ180-226 have been described (Baisden et al., 2001). For transient transfections, the cells were first allowed to grow on cover strips which were coated with a 1: 5 dilution of Matrigel (Becton-Dickinson) for 6 hours. The transfection was carried out using 5 µg DNA using a lipofectin reagent (Invitrogen). The cells were fixed with paraformraldehyde after 60 hours, washed and stained with a monoclonal α-Fyn antibody (Santa Cruz) or an α-AFAP polyclonal antibody from rabbits (Baisden et al., 2001). literature

Baisden., J.M., Qian, Y., Zot, H.M., and Flynn, D.C. (2001). The actin filament-associated protein AFAP-110 is an adapter protein that modulates changes in actin filament integrity. Oncogene 20, 6435-47.

Bergmann, E., Wanzel, M., Weber, A., Shin, I., Christiansen, H., and Eilers, M. (2001) Expression of P27 (KIPl) is prognostic and independent of MYCN amplification in human neuroblastic, int. J. Cancer 95, 176-83.

Bouchard, C, Dittrich, 0., Kiermaier, A., Doh ann, K., Menkel, A., Eilers, M., and Luscher B. (2001). Regulation of cyclin D2 gene expression by the Myc / Max / Mad network: Myc- dependent TRRAP recruit ent and histone acetylation at the cyclin D2 promoter. Genes Development 15, 2042-7.

Callow, M.J., Dudoit, S., Gong, E.L., Speed, T.P., and Rubin, E.M. (2000). Microarray expression profiling identifies genes with altered expression in HDL-deficient mice. Genome Research 10, 2022-9.

Clementi, F., Cabrini, D., Gotti, C, and Sher, E. (1986) Pharmacological characterization of cholinergic receptors in a human neuroblastoma cell line. J. Neurochem 47, 291-7.

Dysvik, B., and Jonassen, I. (2001) J-Express: exploring gene expression data using Java. Bioinformatics 17, 369-370.

Hegde, P., Qi, R., Abernathly, K., Gay, C, Dharap, S., Gaspard, R., Hughes, JE, Snesrud, E., Lee, N., and Quackenbusch, J. (2000). A concise guide to cDNA microarray analysis. Biotechniques 29, 548-50, 552-4, 556 passim.

Pahlmann, S., Odelstad, L., Larsson, E., Grotte, G., and Nilsson, K., (1981). Phenotypic changes of human neuroblastoma cells in culture induced by 12-0-tetradodecanoyl-phorobol-13-acetate. Int J Cancer 28, 583-9.

Westfall, PH, and Young, SS (1993) Resampling-Based Multiple Testing. Examples and Methods for p-Valua Adjustment. Wolf, RM, Wilkes, JJ, Chao, MV, and Resh, MD, (2001). Tyrosine phosphorylation of pl90 RhoGAP by Fyn regulates oligodendrocyte differentiation. J. Neurobiol. 49, 62-78.

Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Hgai, J., and Speed, T.P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Rerearch 30, el5.

Claims

claims

1. Use of modulators of the signal transduction pathway via the protein kinase Fyn for the prophylaxis and / or treatment of tumor diseases.

2. Use of modulators according to claim 1 for the treatment of neuroblastomas.

3. Use according to claim 1 or 2, wherein the modulators are inhibitors of enzymes which are inhibited directly or indirectly by active Fyn.

4. Use of modulators according to claim 1 or 2, which lead to an increase in Fyn activity in the tumor cells.

5. Use of modulators according to claim 1 or 2, wherein the modulators are inhibitors of protein kinase CSK.

6. Use of modulators according to one of claims 1 to 4, wherein the modulators are inhibitors of Rho-Kinase.

Use of modulators according to one of claims 1 to 4, wherein the modulators are inhibitors of MAP phosphatase.

8. Use of modulators according to one of claims 1 to 4, wherein the modulators are activators of protein kinase C.

9. Use of modulators according to claim 1 or 2, which inhibit the proteolytic degradation of Fyn kinase in vivo.

10. Use of modulators according to claim 1 or 2, the modulators being inhibitors of phosphatases which

Counteract Fyn are.

11. Use of modulators according to claim 1 or 2, which contribute to an increase in signal transduction by Fyn.

12. Use of modulators of the signal transduction path via protein kinase Fyn for the manufacture of a medicament for suppressing the metastasis of tumors in the early stages.

13. Use of modulators according to claim 9, wherein the tumor is a pediatric or an adult tumor.

14. Use of modulators according to one of the preceding claims as part of a pharmaceutical composition, characterized in that the composition in addition to the modulator as an active ingredient optionally comprises carriers and / or adjuvants.

15. Use of modulators according to one of the preceding claims, characterized in that the modulator is in the form of a pharmacologically acceptable salt, solvates, hydrates, or a pharmacologically acceptable formulation.

16. Use of modulators according to one of the preceding claims, characterized in that the modulator is present as a prodrug, comprising the modulator and at least one pharmacologically acceptable protective group which is split off under physiological conditions.

17. Use of modulators according to one of the preceding claims, characterized in that the modulator is administered orally, parenterally, rectally, by inhalation, transdermally or intranasally.