WO2003033529A2

WO2003033529A2 - Tr4, tr4 activators, tr4 inhibitors or tr4-associated molecules for treating leukaemic diseases

Info

Publication number: WO2003033529A2
Application number: PCT/EP2002/011484
Authority: WO
Inventors: Martin Zenke; Petr Bartunek; Jaime Madruga; Nicolas P. Koritschoner
Original assignee: Max-Delbrück-Centrum für Molekulare Medizin
Priority date: 2001-10-12
Filing date: 2002-10-14
Publication date: 2003-04-24
Also published as: AU2002362891A1; WO2003033529A3; DE10150183A1

Abstract

The invention relates to the use of TR4, TR4 activators, TR4 inhibitors and/or TR4-associated molecules for the diagnosis, prophylaxis, progression control, treatment and/or follow-up treatment of tumorous diseases and/or diseases of the blood, in particular leukaemia.

Description

Use of TR4, TR4 activators, TR4 inhibitors or TR4-associated molecules for the treatment of leukaemic diseases

The invention relates to the use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules, such as e.g. Antibodies or ligands, and an agent comprising TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the diagnosis and treatment of tumor diseases and blood disorders, in particular leukemic diseases. Fields of application of the invention are the pharmaceutical industry and medicine.

Nuclear receptors are key regulators of cell proliferation, cell differentiation and the various development processes of the cell. In many cases these receptors perform their function by binding to known ligands such as retinoids, steroids, thyroid hormone, eicosanoids and vitamin D. Furthermore, there is an even greater number of nuclear receptors that have no ligands or whose ligands have not yet been identified. These nuclear receptors are also called orphan

Designated receptors (Mangelsdorf and Evans, Cell 83

(1995) 841-850). Numerous studies have shown that nuclear receptors have a strong influence on the

Have development of hematopoietic cells. For example, the retinoid X receptor and the thyroid hormone receptor regulate erytrocyte growth and erytrocyte differentiation (Bartunek et al., Mol. Endocrinol., 12 (1998) 1269-1279). In other cell systems, the activation of RAR by retinoic acid inhibits cell growth and supports the differentiation tion. on neutriphiles (Collins et al., Mol. Cell. Biol. 10 (1990) 2154-2163). TR4 is an orphan receptor that is closely related to the TR2 receptor. Both receptors define a subfamily within the steroid / thyroid receptor family (Hirose et al., Mol. Endocriol., 8 (1994) 1667-1680).

Differentiation and growth of different blood cells is a crucial process in the individual development of more developed organisms, especially mammals. Disorders of the growth and differentiation of blood cells or of blood-forming cells lead to severe disorders in the physiology of the organism, which range from minor metabolic restrictions to the most serious tumor diseases. A typical disease of blood-forming cells can be leukemia, for example. The so-called leukemia cells can, for example, colonize in the bone marrow, usually also in the blood and in lymphatic tissues, but also in all other organs of the organism and lead there to further pathological processes. Various methods are described in the prior art for diagnosing or treating tumor diseases which relate in particular to blood-forming cells. However, the methods and pharmaceutical agents currently available do not permit efficient and reliable diagnosis or therapy of the tumor diseases mentioned.

The object of the invention was therefore to provide methods and means with which tumor diseases, in particular those which relate to the hematopoietic system, can be diagnosed or treated prophylactically, treated and / or treated at an early stage. The invention solves this technical problem by using TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for diagnosis, prophylaxis, follow-up, therapy and / or post-treatment of tumor diseases and / or blood disorders.

For the purposes of the invention, TR4 means an orphan receptor. Accordingly, TR4-associated molecules are, in particular, ligands, antibodies or else markers that bind to the receptor. However, the person skilled in the art is aware that for certain examinations of the orphan receptor, he can use not only the receptor itself, but also the nucleic acid encoding the receptor. For the purposes of the invention, TR4 is therefore understood to mean the receptor both on the nucleic acid and on the amino acid level. TR4 can therefore be the receptor that naturally occurs on or in the cells, but also the nucleic acid that codes for this receptor. Of course, the TR4 receptor can also be synthetically produced receptors or their fragments. The receptor can also be modified biological structures, such as a labeled structure such as HA-TR4 or a modified form such as a TR4 with a mutated DNA binding domain such as ΔTR4. Nucleic acids encoding TR4 are all nucleic acids that code for a structure that is analogous to the receptor and has its biological function. TR4-associated molecules can accordingly be any biological or chemical structure that can interact with TR4 in such a way that an interaction can be detected, such as RXR or modified forms such as ΔRXR. If the coding nucleic acid is used instead of the orphan receptor TR4, for the purposes of the invention, TR4-associated molecules are, for example, promoters or regulatory elements which stimulate or inhibit the expression of the orphan receptor TR4. Under TR4-associated molecules can therefore also be understood as antisense constructs, in particular RNA interference molecules, which suppress the expression of the TR4 orphan receptor. TR4 inhibitors or TR4 activators are all molecules that inhibit or activate the action of the TR4 at the nucleic acid or amino acid level. For example, RIP-140 is a co-activator that interacts with TR4 and can modulate its activity. The skilled worker is familiar with further molecules which can be used as a TR4 activator or TR4 inhibitor. The invention thus relates to the surprising teaching that TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules on the protein or nucleic acid level in diagnosis, prophylaxis, monitoring, therapy and / or in the aftertreatment of tumor diseases and / or blood disorders can be used.

Tumor diseases in the sense of the invention are all pathological changes in cells or cell collections, cell aggregates or tissues which lead to individual cells or cell groups not showing normal growth or differentiation behavior. This means that the cells can multiply uncontrollably, for example; it is also possible that blood stem cells no longer differentiate into mature blood cells, but remain in the stage of the transition from a blood stem cell to a differentiated cell and do not develop into a mature cell. Blood disorders in the sense of the invention can be any deviations from the normal physiological value that affect individual blood cells or blood precursor cells, such as hemolytic disease, hemophilia, bleeding abnormalities and certain forms of leukemia, unless they are associated with the tumor diseases. Blood disorders are accordingly all pathological changes in the erythrocytes or erythrocyte precursor cells Granolocytes, monocytes, lymphocytes and platelets as well as all progenitor cells of these cells as well as all pathological changes in plasma proteins or other components of the serum.

According to the invention it could be shown, inter alia, that the orphan receptor TR4 is surprisingly strongly expressed in hematopoietic cells and tissues and exerts a strong influence on these cells. Accordingly, by detecting the orphan receptor at the protein or nucleic acid level, in particular the DNA or RNA level, it can be diagnosed by comparison with control values of healthy patients whether individual blood or blood precursor cells are in a physiological or pathophysiological Condition. The control or standard values can relate to the concentration or activity of the receptor, the number of nucleic acids encoding it, the concentration or activity or ligands or other parameters. The detection of TR4-associated molecules other than the ligand also allows diagnostic conclusions to be drawn about the condition of individual cells or the tissues which have the receptor and / or its coding nucleic acids. If the person skilled in the art has detected a pathological level of TR4 or TR4-associated molecules, he is able, particularly by genetic engineering or pharmacological interventions, to modify the activity or the concentration of TR4 or TR4-associated molecules in such a way that a physiological or normal or largely healthy condition can be achieved. Such methods are known to the person skilled in the art. According to the invention, it was shown in particular that TR4 supports or induces the proliferation of myeloid progenitor cells; if the person skilled in the art wishes to prevent the proliferation of these cells, in particular in a patient or else in a culture medium, he can selectively use inhibitors or antagonists of the TR4 at the protein or DNA or RNA level to prevent them. If, on the other hand, the person skilled in the art wants to induce the proliferation of myeloid precursor cells in an organism or in a cell culture, he can use, for example, activators which increase the activity of the orphan receptor TR4 or its expression. Such substances are known to the person skilled in the art.

In a particular embodiment of the invention, TR4 comprises a TR4 receptor with a deleted DNA binding domain. In the sense of the invention, this structure is also referred to as ΔTR4. The orphan receptor, which has a deleted DNA binding domain, is advantageously present not only in the cell nucleus, but also in the cytoplasm of TR4-transformed cells, in particular fibroblasts and myeloid cells. TR4 can advantageously be used in the suppression and activation of transcription factors involved in gene expression. This double activity of TR4 is particularly dependent on the TR4 DNA contacts or on TR4 cofactor bonds, it is of course also possible that the activity is dependent on both contacts or bonds. Advantageously, TR4 with a deleted DNA binding domain does not attach to certain TR4 binding sites, such as AGGTCAAAGGTCA. In contrast to TR4, TR4 with the deleted DNA binding domain - ΔTR4 - suppresses the transcriptional activation that depends on RXR or the thyroid hormone receptor-dependent transcriptional activation. For example, ΔTR4 can be used in processes or uses in which the suppression of transcription or binding to the DNA is not desired. is. Surprisingly, ΔTR4 allows myeloid cells to grow. In terms of their morphology and their cell surface marker, TR4 and ΔTR4 are advantageously expression identical. This means that the growth-promoting activity of TR4 is also present in ΔTR4, with ΔTR4 advantageously not suppressing transcription or not binding DNA.

In a further preferred embodiment of the invention, TR4-associated molecules are either ligands, RXR and / or RIP-140. Various ligands, in particular small, low molecular weight molecules, are known to the person skilled in the art and can be used to specifically bind to the receptor TR4. TR4-associated molecules can, however, also preferably be RXR and / or RIP-140. TR4 has a strong repressive effect on RXR-mediated transcription. However, in addition to RXR, it is also possible to use an RXR structure which no longer has a DNA-binding domain or whose DNA-binding domain has been deleted. RIP-140 is a co-activator that interacts with TR4 and can advantageously be used for diagnosis, prophylaxis, follow-up, therapy and / or post-treatment of tumor diseases and / or blood disorders.

In a preferred embodiment, the therapy and / or after-treatment of the tumor disease and / or the diseases of the blood is a differentiation therapy. In the differentiation therapy, in particular non-toxic pharmaceutical compositions can be used to induce or promote a terminal differentiation of, in particular, leukemic cells. Such substances can be, for example, PMA, retinoids, vitamin D, vitamin E, vitamin A, beta-carotene, estrogens, glucocorticoids, interferons, TNF, dimethyl sulfoxides, N-methylformamides and PKC activators or inhibitors. All of these substances can be used together with TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules. be used to treat tumor diseases and / or blood disorders.

In a further preferred embodiment of the invention, TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules are used to produce a medicament for the treatment of tumor diseases and / or blood disorders. In addition to the structures mentioned, the medicament can in particular comprise at least one pharmaceutically acceptable carrier.

In a preferred embodiment of the invention, leukemia is diagnosed and treated as a tumor disease. Preleukemia, acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, secondary leukemia and / or chronic lymphatic leukemia are preferably diagnosed and / or treated as leukemia. Leukemia in the sense of the invention is a collective term for diseases that result from malignant transformation of hematopoietic and / or lymphatic cells, with proliferation and accumulation of neoplastic cells - the so-called leukemia cells - primarily taking place in the bone marrow, but mostly also in the blood and in lymphatic tissues and in other organs. The symptoms result in particular from the displacement of normal blood cells, the impairment of the immune system and the infiltration of atypical cells in organs and lead, among other things, to anemia, bleeding, infections, irritation symptoms, enlargements and functional impairments of affected organs, such as those with meningoencephalomyelopathia leucaemica, which are associated with combined kidney infiltration. Acute leukemia or ^' immature cell leukosis is a disease of the hematopoietic stem cells with proliferation immature Blasts in the bone marrow and mostly also in the blood. Acute lymphoblastic leukemia or acute lymphoblastic leukemia is the most common childhood leukemia, the pathogenesis of which is characterized by a malignant transformation of a lymphatically determined stem cell. In the sense of the invention, acute lymphoblastic leukemia is also understood to mean lymphoblastic non-Hodgkin's disease. Acute myeloid leukemia or acute non-lymphatic leukemia in the sense of the invention is leukemia which affects approximately 80% of all acute leukemia in adulthood and is the second most common childhood leukemia. This disease is induced by the malignant transformation of a myeloidally determined stem cell. Acute undifferentiated leukemia is a rarely diagnosed form to which most leukemias can be assigned in the sense of the invention. Chronic lymphoblastic leukemia is a common chronic leukemia that has a low degree of malignancy and is characterized by enlargement of the spleen and liver. Chronic myeloid leukemia is middle-aged leukemia, whereby in the sense of the invention the acquired Philadelphia chromosome is detectable in most cases. Chronic myelomonocytic leukemia is a stem cell disease belonging to the myelodysplastic syndromes. Smoldering leukemia is the slow course of acute - mostly myeloid - leukemia, which can be demonstrated by a low DNA-metabolism in the bone marrow cells. For the purposes of the invention, secondary leukemia is a term which is not used uniformly, since secondary leukemia can arise as a result of other hematopoietic stem cell diseases, in particular myelodysplasias. Accordingly, all forms of leukemia due to toxic influences are secondary leukemia in the sense of the invention. In addition to the use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules, Treatment of the above-mentioned leukemias, intrathecal chemotherapy, adequate supportive measures such as blood cell replacement, infection prophylaxis and treatment are also used. Treatment with cytostatics or a bone marrow transplant is also possible. Of course, the patients in question can also be treated by a splenectomy, optionally with gamma globulins, anthrax irradiation, administration of cytostatics and interferons and similar therapy schemes known to the person skilled in the art.

The invention also relates to the use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the proliferation and / or differentiation of blood cells, pluripotent and / or determined stem cells. TR4, ΔTR4, or TR4 activators, inhibitors and / or TR4-associated molecules can be used for the proliferation, differentiation and / or outgrowth of various cells, in particular blood cells and blood precursor cells.

In a further advantageous embodiment of the invention, TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules are used to proliferate blood cells or pluripotent and / or determined stem cells or to induce outgrowth of these cells.

In a further preferred embodiment of the invention, TR4; TR4 activators, TR4 inhibitors and / or TR4-associated molecules are used to prevent terminal differentiation of blood cells, hematopoietic cells, pluripotent and / or determined stem cells. The invention also relates to the use of TR4, TR4 activators and / or TR4-associated molecules for the production of myeloid progenitor cells. Myeloid progenitor cells are all cells that can develop into megakaryocytes, thrombocytes, eosinophils, neutrophils, basophils, mast cells, macrophages, monocytes and antigen-presenting cells.

The invention also relates to the use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the screening of medicaments. Through the teaching according to the invention and through the detection according to the invention of the high proportion of TR4 or of TR4-associated molecules in pluripotent, determined stem cells and / or in hematopoietic cells, it is possible for the person skilled in the art to screen drugs in a targeted manner that use TR4 or TR4 - Associated molecules, in particular ligands of TR4, interact. Those skilled in the art are aware that they can use all combinatorial techniques to screen drugs that can interact with TR4 or with TR-associated molecules. For this purpose, for example, the methods of multiple peptide synthesis, nanosynthesis reactions or free and polymer-bound polymer libraries or low-molecular libraries can be used. It is also possible to use the phage display method, combinatorial mutagenesis or in vitro protein synthesis to analyze libraries in order to detect structures that interact with TR4 or TR4-associated molecules as a potential drug. Basic work on generating libraries is also known to the person skilled in the art, such as, for example, pin synthesis, the process of dividing-mixing, dividing-coupling-reuniting, the synthesis of one peptide per grain, the parallel-chemical synthesis and / or spot synthesis. The identified product can then be Synthesis such as microsequencing or mass spectrometry, affinity selection, chemical coding or otherwise determined.

The invention also relates to the use of TR4 and / or TR4-associated molecules for identifying or isolating a TR4 antagonist or activator or agonist, the potential activator or antagonist being brought into contact with the TR4 and / or TR4-associated molecules and the binding, activation or inhibition of the TR4 or the TR4-associated molecules is determined. After TR4 has been detected as an orphan receptor on or in hematopoietic cells according to the invention, it is possible for a person skilled in the art to determine which ligands or antagonists or activators can bind to TR4 and a physiological one with the aid of association, interaction or binding tests or induce pathophysiological signal chain.

The invention . also relates to a pharmaceutical composition comprising TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules, optionally with a pharmaceutically acceptable carrier. In particular, the activity of the TR4 receptor can be influenced with the composition.

The invention also relates to a diagnostic kit comprising TR4, TR4 activators, TR4 inhibitors and / or TR-associated molecules.

The invention is to be explained in more detail below using an example, without being restricted to this example. example

Cells and cell culture

Chicken marrow cells from 3 to 10 day old SPAFAS chickens were prepared and the cells cultured. For this purpose, the bone marrow cells were grown for 2 to 4 hours in standard nutrient medium consisting of DMEM, 8% fetal calf serum, 2% chicken serum, 20 mM HEPES (pH 7.3) and 100 units penicillin / streptomycin, standard nutrient medium. The non-adherent cells were then processed and used for infection and the infected cells in DMEM, 8% FCS, 5% chicken serum, 0.128 mg / ml chicken transferrin (conalbumin), 100 ng / ml recombinant chicken stem cell factor, 20 mM HEPES (pH 7.3) and 100 units penicillin / streptomycin were cultivated at a cell density of 2 to 3 x 10 ⁶ cells / ml.

The chicken embryo fibroblasts were grown in the standard growth medium. The virus-producing chicken embryo fibroblasts were prepared and the bone marrow cells were infected using standard methods. The infections were carried out in the presence of 1 μg / ml recombinant human insulin and 100 ng / ml stem cell factor.

Construction of retroviral vectors

A retroviral vector was constructed which contained the entire strand length of the TR4-CDNA down along the CMV promoter. Wild-type Human-TR4 was installed at the EcoRI interface of pSFCV-LE. The TR4 versions labeled with haagglutinin were first converted to a carrier vector which contained coding sequences for three hemagglutinin epitopes upwards along the clone sites. In the HA-ΔTR4 amino acid, positions 64 to 232, which encompass the entire DNA binding domain of human TR4, were removed. Then the EcoRV-EcoRI- Locations of pSFCV-LE HA-TR4 and HA-ΔTR4 installed. Recombinant retroviral vectors, the RXR and -ΔRXR freed from its DNA binding domain are already state of the art.

cDNA synthesis and PCR

RNA production, cDNA synthesis and PCR amplification are carried out according to the standard procedures. For this purpose, a total of 20 to 50 μl of strand cDNA was first synthesized from 2-5 μg of total RNA using 100 U M-MLV revertase and random hexamer primers. For PCR amplification, a set of degenerate primers was developed for the area encoding the DNA binding domain of the nuclear receptors. These primers had the following sequences:

NHRl 5 '-GAYRARDCNWSNGGNTDYCAYT-3

NHR2: 5 '-GAYMGNDSNWSNGGNAARCAYT-3';

NHR3: 5 '-CAYTTNYKNWRNCKRCANKMNKGRCA-3';

NHR4: 5 '-TGYAARNNNTTYTTYMRNMG-3'. The strand cDNA was first subjected to 40 cycles of PCR amplification using Taq DNA polymerase and with the following cycle profile: 30 seconds at 94 ° C., 90 seconds at 37 ° C. and 20 seconds at 72 ° C. The following primer combinations were used: In the first reaction, NHR1 or NHR2 together with NHR3. The product of this reaction was diluted 1: 300 and used as a matrix for the reaction with NHR3 and NHR4. The PCR products were purified by gel electrophoresis and incorporated into the vector pCRII. The resulting clones were sequenced and then the sequences were analyzed using the BLAST tool to identify functional units. The Genbank accession number for the Chicken TR4 is AF133054. RNA analysis

The RNA was isolated from tissue and the following cells using standard procedures: Human cells: K562 (Erythroid); U937, HL60 (myeloid); Namalwa, Raj i (B lymphoid); CEM, Jurkat (T-lymphoid) and HepG2 (liver). Mouse: MEL, E31 (erythroid); WEHI-3, RAW (myeloid); A20 (B lymphoid) and NIH3T3 (fibroblast). Chicken: DT40 (B lymphoid); MSB-1 (T-lymphoid) as well as v-relER-converted progenitors and dendritic cells. The following primary cells were used: human cells: erythroid rogenitors, peripheral blood T cells and monocytes; Mouse cells: dendritic cells; Chicken cells: SCF and STED erythroblasts. 15 μg RNA were analyzed by Northern blot. For this purpose, the filters were hybridized with ³² P-labeled human or chicken TR4-specific probes, washed moderately intensively and photographed.

Flow cytometry

Cells were collected for flow cytometry, washed with PBS (1% BSA) and incubated with primary antibodies (1 h). The following monoclonal antibodies were used: MC47-83, MC51-2, MC22-3; Kl, 2G11; JS8 and JS4, CD3, CD4 and CD8; MEP17, MEP21, MEP26 and EOS47; DM-GRASP (BEN1); HEM-CAM antibody; CD44 (AV6). The cells were then incubated with FITC-conjugated anti-mouse IgG for one hour, washed and slurried in PBS (1% BSA, 2 μg / ml propidium iodide) in order to allow the viable cells to emerge. These cells were analyzed using flow cytometry.

Western blot

The entire cell extracts were prepared on a 10 percent SDS-PAGE gel and blotted on nitrocellulose membranes. The following antibodies were used: anti-hemagglutinin 12CA5, anti-CEBPß, anti-Mim-I. The blots were then washed and incubated with the corresponding secondary antibody for 45 minutes in PBS (5% non-fat milk powder) at room temperature, developed and photographed.

immunofluorescence

The chicken embryo fibroblasts containing the TR4 versions were cultured as above and trypsin was added. They were then given the opportunity to adhere to glass slides (1 h, 37 ° C). The cells were washed and fixed with PBS (3.7% paraformaldehyde) for 15 minutes. After fixation, the cells were made permeable with PBS (0.5% NP-40) (15 min), incubated with 0.5% thermally inactivated FCS in PBS for 30 minutes to block possible non-specific bindings, and Reacted with anti-hemagglutinin 12CA5 for 1 h, followed by reaction with FITC-conjugated anti-mouse IgG (1 h). In addition, the samples were stained with TRITC-labeled phalloidin for actin. DAPI (0.5 mg / ml) was used to stain the cell nuclei. The samples were washed with PBS and fixed in Mowiol 4.88 containing 50 mg / ml DABCO as a bleach protection agent. All incubations were carried out at room temperature.

Protein purification and sequencing

The protein bands stained with Coomassie blue were removed and digested with trypsin in the gel. Trypsin peptide maps were created using the reversed phase HPLC (μRPC C2 / C18 SC 2nd 1/10 column). The flow rate was 100 μl / min at 25 ° C using a linear acetonitrile gradient in 0.1% trifluoroacetic acid. The peptides of interest were placed on a glass filter fiber of a procise- coated with Biobrene Sequenators applied. The subsequent sequencing was carried out using standard protocols.

Band Shift Assays The chicken embryo fibroblasts were dissolved in a buffer solution of 20 mM Tris pH 7.5, 2 mM DTT, 20% glycerol and 400 mM KCl - (1 h on ice). The complete cell extracts (5 - 10 μg) were incubated for 15 minutes on ice with a ³² P-labeled oligonucleotide which is the DRI-HRE binding site

5 '-GGGGATCCGGCAGAGGTCAAAGGTCAAACGT-3' contained. Poly-dl-dC (1 μg per reaction) was added as a non-specific competitive reagent. A mutated version of the DRI-HRE position was used for competitive experiments

5 '-GGGGATCCGGCAGTGGACAATGGACAAACGT-3'

used. The binding reactions were separated on 5 percent polyacrylamide gel in 0.25x TBE.

Transient transfections

By means of calcium phosphate precipitation, the chicken embryo fibroblasts were transiently transfected with retroviral expression vectors which contained TR4, HA-TR4 and HA-ΔTR4, in combination with eukaryotic expression vectors which carried RXR in pSG5, and a luciferase reporter with rat CRBPII-RXRE. In order to normalize the transfection effectiveness, RSV-ß-galactose and pCHHO plasmids were also cotransfected. The transfected cells were grown in a standard nutrient medium which largely contained FCS and chicken serum extracted from retinoids. 9cRA (10 ^"6 M) was added. Forty-eight hours later, the cell extracts were prepared and for their luciferase and β-galactosidase activity analyzed. The luciferase values were normalized to investigate the β-galactosidase activity.

It has been found that the orphan receptor TR4 is surprisingly strongly expressed in hematopoietic cells and tissues and exerts a strong influence on these cells. Furthermore, it was found that TR4 ectopically expressed in the bone marrow by means of a retrovirus vector promotes the proliferation of myeloid progenitor cells. Another finding is that the activity of TR4 does not depend solely on its association with DNA, because the expression of a mutated version of TR4 without the possibility of DNA binding leads to the same proliferative potential as with wild-type TR4.

Overall, it emerges from these investigations that the orphan receptor TR4 is an important regulator of the proliferation and development of myeloid progenitor cells.

In order to determine the expression profile of nuclear receptors in hematopoietic cells, a strategy based on RT-PCR was used which made use of degenerate primers with the DNA binding domain of the nuclear receptors, which was largely preserved. It has been found that several members of the nuclear receptor gene family occur in different types of progenitor derived from chicken bone marrow. Interestingly, one of the cDNA clones most present in the studies on the invention corresponded to the chicken homologue of the nuclear orphan receptor TR. In order to investigate the expression pattern of TR4 in more detail, human and chicken TR4 CDNA were used as probes in Northern blots which contained an RNA field from hematopoietic and non-hematopoietic tissues and cell types (FIGS. 1A, B and C). A striking TR4 expression was found in human myeloid cell lines (U937, HL60) and primary monocytes and dendritic cells (Fig. 1A). TR4 was similarly pronounced in rat myeloid cells (Fig. 1B). Moderate was found in the T cells and only a little TR4 in the B cells. In addition, TR4 was immediately detectable in human, mouse and chicken erythroid cell lines (K562, E31 and HD3) and in the primary progenitors of red blood cells (Figs. 1A, B and C). As expected, there was a high level of TR4 expression in the brain. The TR4 level was also high in the thymus and somewhat lower in bursa and spleen (Fig. IC). Accordingly, TR4 transcripts were found in chicken B, T and dendritic cells. As a result, TR4 is present in a number of different cell types in the haematopoietic system.

TR4 effectively induces the proliferation of myeloid progenitor cells.

While previous studies have shown the importance of TR4 for the suppression of various NR pathways, the influence of TR4 on primary cells, especially the hematopoietic system, has not yet been investigated. Consequently, in order to gain an insight into the potential role of TR4 for the hematopoietic cells, TR4 was ectopically expressed in chicken bone marrow cells and the effects of the receptor on the proliferation or development of these cells were investigated. For this purpose, a retroviral vector carrying TR4-CDNA was constructed and transfected into the chicken embryo fibroblasts and the neomycin-resistant cells were selected. The cells clearly showed the TR4 protein. (Fig. 3) and were used as a source for infection of the bone marrow cells with the virus. With pilot experiments in which different cultural conditions with different Different combinations of growth factors, hormones, sera and conditioned media were used, the growth of the progenitor cells infected with TR4 was optimized. The most striking observation was that TR4 caused a cell outgrowth within 4 to 5 days, which could not be found in the reference cultures infected with an empty vector (Fig. 2). The cells grew in large aggregates consisting of several hundreds of cells (Fig. 2). This proliferation stopped after 14 to 16 days.

Histological examination of the TR4 cells showed a similarity to myeloid progenitors (Fig. 2). The cells had an average diameter of 9 μ and were further characterized by flow cytometry using a panel of monoclonal antibodies with regard to the appearance of cell surface markers (Fig. 2). A high expression of the myeloid markers MC47-83, MC51-2 and MC22-3 and MHC class II was found in the TR4 cells. The test for Kl, a macrophage marker, was negative. Using antibody JS8, a transferrin receptor was found as expected (since the cells were in the exponential growth phase). However, the TR4 cells had neither the T cell-specific marker (CD3, CD4 and CD8) nor the erythroid-specific marker (JS4) or the eosinophilic marker (EOS47; Fig. 2). Likewise, there was no HEM-CAM, an adhesion molecule from earlier hematopoietic progenitors, whereas CD44, another adhesion molecule in the form of different hematopoietic cell types, was detected. Furthermore, MEP17 / α2 / ßl integrin was found in the TR4 cells, while MEP21 / thrombomucin, which occurs to a large extent on platelets and multipotent myeloid progenitors, platelet and erythroid cells, was missing. It can be concluded that TR4 cells are arky myeloid progenitors due to their morphological properties and the profile of their cell surfaces.

The growth-promoting activity of TR4 is impaired, but not eliminated, when its DNA binding domain is deleted.

TR4 has been described as a transcription factor involved in the suppression and activation of gene expression. This double activity can depend significantly on the TR4 DNA contacts or TR4 cofactor (s) linkages, or both. In order to investigate which activity of TR4 could be involved in the growth promotion of bone marrow cells observed here, retroviruses which contained full-length TR versions labeled with hemagglutinin or mutant TR4 without its DNA binding were therefore constructed and analyzed (Fig. 3 ). Western blot using an anti

Hemagglutinin-specific antibody has been an effective one

Expression of the HA-TR4 and HA-ΔTR4 proteins found

(Fig.3B). In addition, the subcellular localization of the TR4 proteins was determined by indirect immunofluorescence (Fig. 3). In the chicken embryo fibroblasts, HA-TR4 was clearly restricted to the cell nucleus, while the HA-ΔTR4 protein appeared both in the cell nucleus and in the cytoplasm. The latter observation is not surprising, since TR4 and its closest "relative" TR2 share a highly homologous nuclear localization signal in their DNA binding domain and it is precisely this motif that was removed in HA-ΔTR4. As expected, the staining of chicken embryo fibroblasts containing empty vectors with hemagglutinin-specific antibodies was negative. Using band shift assays, it was demonstrated that HA-TR4 had effectively attached to the TR4 binding site contained in DR1-HRE (AGGTCAAAGGTCA), whereas HA-ΔTR4 had not (Fig. 4). The band corresponding to HA-TR4 was shifted slightly more than with the unmarked TR4. The HA-TR4 binding was clearly specific because it was competed effectively by unlabeled DR1-HRE but not by a mutated oligonucleotide (Fig. 4). In addition, the anti-hemagglutinin-specific monoclonal antibody was able to cause a supershift of the TR4-HA band, whereas it had no influence on the pattern of the HA-ΔTR4. These data were further confirmed by shift western experiments in which the proteins in the shift gel were transferred to a membrane and probed with the anti-hemagglutinin antibody. As expected, the antibody recognized HA-TR4, which corresponded to the band subjected to a shift.

Transient transfection experiments were carried out to analyze the transcriptional activity of TR4 constructs. It was found that HA-TR4 suppressed RXR- and thyroid hormone receptor-dependent transcriptional activation, HA-ΔTR4 did not (Fig. 4). Similar results were obtained from hematopoietic cells (HD3 erythroblasts).

After determining that HA-ΔTR4 neither binds the DNA nor suppresses the transcription, the next step should be to investigate whether the TR4-induced phenotype is responsible for the DNA binding activity of TR4. HA-TR4 and HA-ΔTR4 retroviruses were used to infect bone marrow cells as described above. HA-TR4 behaved identically to unmodified TR4, so that myeloid cells grew without problems were obtained. Surprisingly, growth of myeloid cells was also observed in HA-ΔTR4, although the total number of cells was somewhat lower than in HA-TR4. As expected, the infected with a Neovirus control cells showed limited growth potential and completed the proliferation after 4 to 6 days ^• in the culture. In addition, the cells converted with retroviruses clearly showed hemagglutinin-labeled TR4 proteins, as was shown by a Western blot analysis using the anti-hemagglutinin-specific antibody. Finally, it was found that TR4, HA-TR4 and HA-ΔTR4 are identical in terms of both their morphology and their cell surface marker expression.

In conclusion, it can be concluded that TR4 and, to a lesser extent, HA-ΔTR4 effectively support outgrowth of myeloid cells from bone marrow, which shows that the growth-promoting activity of TR4 is not lost through the deletion of its DNA-binding domain.

RXR and its DNA-binding variant ΔRXR only partially repeat the TR4 effects on bone marrow cells.

As already described above, TR4 has a strong repressive effect on RXR-mediated transcriptions. Therefore, a ΔRXR that no longer has a DNA binding domain was introduced into bone marrow cells via a retrovirus in order to check whether a dominant interfering RXR would develop a phenotype similar to TR4. A full strand RXR was used in a control culture. While the ΔRXR and RXR samples initially showed an outgrowth, these cells stopped proliferation on the 9th day in the culture and from then on the cell numbers remained constant or began to decrease. All of this data indicates that extended proliferation is only achieved by TR4 and TR4 variants, whereas RXR promotes the growth of myeloid cells. In addition, ΔRXR showed a similar effect on bone marrow cells as full-length RXR, which shows that, as with TR4, RXR DNA binding is entirely unnecessary for this activity.

To investigate the differentiation potential of the TR4 cells, myeloid differentiation inducers such as 9-cis-retinoic acid or TFA were added, and the cells were then examined for morphological changes and their adhesion properties. As a control, further cells were treated with the chicken myelomonocyte growth factor (cMGF), with which a growth-promoting but no differentiating activity was detected in previous experiments. Only a few of the HA-TR4 and HA-ΔTR4 cells treated with 9cisRA became adherent, while the majority of the RXR and ΔRXR cells adhered to the vessel and differentiated into macrophages.

TPA rapidly initiated adhesion of the entire cell population, with no apparent differences observed between RXR, ΔRXR, TR4 and ΔTR4 cells. Different concentrations of TPA were added to the cells and the morphological changes taking place were observed under the microscope. After prolonged TPA treatment (3 days), some of the cells acquired a multicellular nucleus morphology.

TR4 and ΔTR4 cells have high levels of C / EBPß and the promyelocyte marker Mim-1. In the next step the protein expression of the TR4 cells was analyzed. Interestingly, both HA-TR4 and HA-ΔTR4 cells had an abundant protein with a molecular mass of 35 kDa. This band was also observed in RXR and ΔRXR cells, but to a much lesser extent than in the TR4 cells. The identity of this protein was then determined. For this purpose, this band was isolated from a gel and peptide sequencing was carried out. By analyzing different peptide sequences, the protein was identified as the myb-induced myeloid protein 1, called Mim-1. A Western blot with an anti-Mim-1-specific antibody additionally confirmed the identity of this protein. The 18 kDA band found in the Coomassie blue stained gels is most likely a Mim-1 degradation product because it could also be stained with anti-Mim-1 antibody. Mim-1 occurs in cytoplasmic granules in the form of normal and deformed promyelocytes.

In order to further deepen the analysis of the TR4 cells, the presence of C / EBPß, another protein which occurs to a large extent in myeloid progenitors and is required for effective Mim-1 expression, was checked by immunoblot. It was found that cells containing TR4 and HA-ΔTR4 had high C / EBPß. C / EBPß also occurred in RXR and ΔRXR cells, but to a lesser extent.

All in all, all these data confirm the effect that TR4 plays a role in the initiation of outgrowth of bound granulocyte progenitors or in the prevention of terminal differentiation of these cells.

Expression is for understanding how the various hematopoietic lineages develop of nuclear hormone receptors of central importance. The invention demonstrates that TR4 occurs to a high degree in hematopoietic cells and tissues of vertebrate organisms, and particularly strongly in myeloid cells. Furthermore, the influence of TR4 on hematopoiesis was revealed by the ectopic expression of TR4 in bone marrow cells. It was found that TR4 leads to an effective outgrowth of promyelocytes and is thus involved in the proliferation and differentiation of myeloid progenitor cells. In addition, it was shown that TR4 as well as ΔTR4, which has been freed from its DNA-binding domain, have essentially the same biological activities. Obviously, the DNA binding activity of TR4 is not required for its activities in hamatopoetic cells.

As shown here, TR4 occurs in different hematopoietic lineages, but an effective outgrowth after ectopic expression in bone marrow cells was only observed in the myeloid area. Interestingly, although erythroid cells showed striking TR4 expression in all analyzed species, no growth of erythroid progenitor cells was observed. Furthermore, it was found that TR4 in particular has a slight growth-promoting activity when incorporated into existing erythroid progenitors.

According to the invention, the transcriptional activity of TR4 as a repressor is caused by various mechanisms, including the composition of the target site. Accordingly, TR4-mediated repression at certain DNA elements requires an intact DNA binding domain, as was also shown here. Alternatively, transcriptional repression could be carried out by sequestering cofactors (transrepression) which, for example, involve AF-2 connect, be mediated and the transrepression consequently be carried out both by TR4 and ΔTR4. Interestingly, TR4 and ΔTR4 show similar biological activities in promoting promyelocyte proliferation in vivo. This allows the conclusion that the activity of the ectopic TR4 in myeloid cells results largely, if not completely, from the sequestration of one or more cofactors involved in the correct development of the myeloid. This idea is further supported by evidence obtained in experiments with other hematopoietic cell types. For example, the estrogen receptor in erythroid cells can inhibit differentiation through a mechanism that requires an intact AF-2 domain but is independent of the AF-1 and DNA binding domains.

It has recently been shown that NR are able to modulate target gene expression not only as DNA-bound factors, but also through mechanisms that do not require direct DNA interactions. In most cases, such DNA-independent activities of the NR are carried out via protein-rotein interactions. The most likely candidates for such a function are corepressors and coactivators known to interact with the AF-2 domain of NR. Such a DNA-independent mechanism of TR4 activity would correspond to the observations made, which demonstrate a similar activity of TR4 and ΔTR4 in promyelocyte proliferation. A candidate for this role is RIP-140, a proven co-activator that interacts with TR4. Furthermore, transrepression has priority due to RIP-140 sequestration and has been observed, for example, in PPAR / RXR-controlled gene expression. The investigation of the subcellular localization of the TR4 proteins showed that TR4 is present in the cell nucleus, whereas ΔTR4 is mostly in the cell nucleus, but also in the cytoplasm of TR4-transformed chicken embryo fibroblasts and myeloid cells. One reason for this could be that the putative nuclear localization signal from TR4 (like the corresponding nuclear localization signal in the DNA binding region of TR2) is removed in ΔTR4. As a result, the reduced cell nucleus expression of ΔTR4 could well explain the somewhat reduced proliferation potential of the ΔTR4 cells compared to TR4 cells.

Finally, it is demonstrated that TR4 as a regulator of myeloid development is a goal of pharmacological research and thus opens up new possibilities for the treatment of myeloid leukemia.

The illustrations show:

Fig. 1 .: Ha atopoetic cells and tissues show TR4

The total RNA of various cell types and tissues from humans (A), mice (B) and chickens (C) was analyzed for TR4 expression by Northern blot. TR4-specific transcripts are marked (arrowhead and star). 28S ribosome RNA stained with methylene blue is shown to demonstrate the RNA load. Erbls: erythroblasts, DC: dendritic cells (Dendritic Cells), DCprog. : DC progenitors (SC progenitors), SCF and STED: SCF or SCF / TGFα-dependent erythroblasts.

Fig. 2 .: TR4 promotes the proliferation of bone marrow cells in vitro.

Growth curves of bone marrow cells infected with HA-TR4, HA-ΔTR4, RXR and ΔRXR as indicated. Control cells with empty neovirus. The cells were made in a medium 100 ng / ml SCR grown. The total number of lines was determined daily. Small picture: HA-TR4 and HA-ΔTR4 cells effectively show TR4 proteins according to detection by Western blot and anti-hemagglutinin-specific antibody.

Fig. 3 .: DNA binding activity and transcriptional activity of HA-TR4 and HA-ΔTR4.

(A) The DNA binding activity of TR4 proteins was analyzed by band shift assays using complete cell extracts from the stably transfected chicken embryo fibroblasts shown in Fig. 3B. A ³² P-labeled oligonucleotide containing a DRI motif was used for this. The chicken embryo fibroblasts (control) and neoresistant chicken embryo fibroblasts with empty vectors (neoR) showed some endogenous binding activity (lanes 2 and 3, respectively). The HA-TR4 extracts showed a predominant band (arrowhead in the left field, lanes 4 and 6) that was missing in the HA-ΔTR4 samples (lane 7). TR4 favored a slightly faster complex migration compared to HA-TR4 (lanes 8 and 11). When anti-HA-specific antibody (α-HA) was added, the HA-TR4 band was supershifted (marked with an asterisk), whereas HA-ΔTR4 and TR4 remained unchanged. Lane 1: no extract (none). Lane 5: Competition with a 100-fold excess of unlabeled oligonucleotides. Open arrowheads: background bands.

(B) HA-TR4 and HA-ΔTR4 were analyzed for their transcriptional activity by means of transient co-transfection experiments in chicken embryo fibroblasts. The luciferase reporter construct contains rat CRBPII-RXRE. In the transfection reactions, 500 ng of both reporter and pSG5-RXR plasmids (all samples) and increasing amounts (5, 10, 50, 100 and 500 ng) of retrovirus vectors with HA-TR4 or HA-ΔTR4 such as stated used. Luciferase activity was normalized by cotransfection of a ß-galactosidase reporter plasmid. As indicated, 9cRA was added to the solution at a final concentration of 10 ^"6 M (+).

Fig. 4 .: Differentiation of TR4 and RXR cells in response to various stimuli.

The differentiation of the HA-TR4, HA-ΔTR4, RXR and ΔRXR cells on the 12th day in the culture was triggered with 9cRA (lxlO ^"6 M) or by TPA treatment (1 μg / ml) for 48 h The differentiated adherent cells were then stained with a histological dye (DiffQuik). As a control, chicken myelomonocyte growth factor (40 ng / ml) was added. Since bone marrow cells infected with empty neoviruses had no outgrowth, the cells infected with empty vectors could not The density of the adherent cells was determined using NIH imaging software and the values of the cells prepared with TPA were arbitrarily set to 100.

Claims

claims

1. Use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for diagnosis, prophylaxis, monitoring, therapy and / or post-treatment of tumor diseases and / or blood disorders.

2. Use according to claim 1, characterized in that at least one TR4 with a deleted DNA binding domain is used.

3. Use according to claim 1 or 2, characterized in that the R4R and / or RIP-140 molecules associated with TR4 are used.

4. Use according to one of claims 1 to 3, characterized in that the therapy is a differentiation therapy.

5. Use according to one of the preceding claims 1 to 4, characterized in that as a tumor disease

Leukemia is diagnosed and / or treated.

6. Use according to one of the preceding claims 1 to 5, characterized in that as leukemia a pre-leukemia, an acute leukemia, an acute lymphoblastic leukemia, an acute myeloid leukemia, an acute undifferentiated leukemia, a chronic lymphatic leukemia, one chronic myeloid leukemia, chronic myelomonocytic leukemia, smoldering leukemia, a secondary leukemia and / or chronic lymphoblastic leukemia are diagnosed and / or treated.

7. Use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the proliferation, differentiation and / or outgrowth of hematopoietic cells, blood cells, pluripotent and / or determined stem cells.

8. Use according to claim 7, characterized in that TR4 is used to prevent terminal differentiation.

9. Use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the production of myeloid progenitor cells.

10. Use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for screening pharmaceutical agents.

11. Use of TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules for the manufacture of a medicament for the treatment of tumor diseases and / or blood disorders.

12. Use of TR4 and / or TR4-associated molecules to identify and / or isolate a TR4 antagonist or TR4 agonist, characterized in that the potential TR4 agonist or the TR4 antagonist with the TR4 or the TR4- Associated molecule is contacted and the binding, activation or inhibition of the TR4 and / or the TR4-associated molecules are determined.

13. Pharmaceutical composition comprising TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules and a pharmaceutically acceptable carrier.

14. Diagnostic kit comprising TR4, TR4 activators, TR4 inhibitors and / or TR4-associated molecules.