WO2007142139A1

WO2007142139A1 - Method of identifying candidate for remedy of acute myeloid leukemia

Info

Publication number: WO2007142139A1
Application number: PCT/JP2007/061190
Authority: WO
Inventors: Tomoki Naoe; Fumihiko Hayakawa; Mitsunori Okamoto
Original assignee: National University Corporation Nagoya University
Priority date: 2006-06-02
Filing date: 2007-06-01
Publication date: 2007-12-13
Also published as: JPWO2007142139A1; JP5246776B2

Abstract

It is intended to disclose a method of identifying a candidate for a remedy of acute myeloid leukemia which comprises contacting activated mutant FLT3 with Lyn in the presence of a test substance and examining whether or not the test substance inhibits the binding of activated mutant FLT3 to Lyn and/or whether or not the test substance inhibits the phosphorylation of Lyn in a cell expressing activated mutant FLT3. It is also intended to disclose a method of identifying a candidate for a remedy of acute myeloid leukemia which comprises contacting a test substance with a cell expressing Lyn and examining whether or not the test substance inhibits the expression or Lyn and/or whether or not the test substance inhibits the phosphorylation of STAT5 by Lyn. A substance identified by the above method is useful as the active ingredient of a remedy for acute myeloid leukemia.

Description

Method for identifying candidate substances for therapeutic agents for acute myeloid leukemia

Technical field

[0001] The present invention relates to a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, and a therapeutic agent for acute myeloid leukemia.

Background art

[0002] Fms-like tyrosine kinase 3 (FLT3) is a receptor belonging to the type III receptor thymosine kinase as well as KIT, FMS, PD GF receptors and the like. The ligand is thought to be important for the proliferation and differentiation of hematopoietic stem cells because it is expressed in hemocyte pluripotent stem cells and the knockout mice show a decrease in hematopoietic progenitor cells. ing. When the ligand binds to the receptor, the receptor forms a dimer through the ligand, which causes the receptor to autophosphate and activates downstream signaling factors. Signals from FLT3 play a very important role in hematopoiesis by controlling the proliferation of stem cells.

[0003] Recently, the active mutation FLT3 was frequently observed in adult acute myeloid leukemia (AML) cases. In the presence of the active mutant FLT3, the receptor and its downstream signaling factor are constitutively activated and cell proliferation is promoted. As the active variant FLT3, the FLT3 paramembrane region (JMD) gene has an internal tand em duplication (ITD) type (FLT3 / ITD), and the tyrosine kinase domain has a point mutation. (FLT3 / TKD) (Yamamoto Y, et al., (2001). Blood, 97, 243 4-9) is known. FLT3 / ITD mutations have been reported in 20% of AML cases (Yokota, S., et al., (1997) 丄 eukemia, ll, 1605-9), part of overlapping amino acid sequences. (Kiyoi H., et al., (1998) .Leuk emia, 12, 1333-7; Hayakawa F., et al., (2000). Oncogene, 19, 624-31. ). It has been shown in animal models that abnormalities in signal transduction caused by the active mutation FLT3 are involved in the onset and progression of leukemia as first hit or second hit. In addition, the presence of the active mutant FLT3 is a poor prognostic factor for AML, and it is a disease of AML (Frohling, S., et al., (2002) .Blood, 100,4372—80; Gilliland, DG & Griffin, JD (2002) .Blood, 100,1532—42; Horiik e'S.'et al., (1997) 丄 eukemia, ll, 1442-6; Kiyoi, H., et al., (1999) .Blood, 93, 3074-80;

Kiyoi, H., et al., (2005). Int J Hematol, 82, 85—92; Kottaridis, PD, et al., (2001). Bl ood, 98, 1752-9; Nakano, Y., et al., (1999) .Br J Haematol, 104,659-64; Schnittger, S., et al., (2002) .Blood, 100,59-66; Shih, LY, et al., (2004) 丄 eukemia, 18,466- 75; S hih, LY, et al, (2002) .Blood, 100,2387-92) 0 abnormalities of FLT3 signaling these reasons onset of leukemia, plays an important role in the progression, believed Ru RU

[0004] The present inventors first introduced FLT3 / ITD into 32D and BA / F3, which are cell lines showing IL-3-dependent proliferation, and FLT3 / ITD was proliferated in an IL-3-independent manner. (Hayakawa, F., et al., (2000). Oncogene, 19,624-31) _{0 In} particular, STAT5 activation is wild-type FLT3. This is a phenomenon that is not observed in cells that have been introduced, and it is strongly suggested that this may be responsible for IL-3 independent proliferation, but the signaling pathway that links mutant FLT3 and STAT5. I understand! / ,!

[0005] Robinson et al. (Robinson, LJ, et al., (2005). Exp Hematol, 33, 469-79), Lyn is a member of Src family kinases (SFK) following FLT3 ligand stimulation in cells expressing wild type FLT3. Report that is phosphorylated. It also shows abnormal phosphorylation of SFK in cell lines expressing FLT3 mutant, but due to the difference in the experimental system from that used for wild-type FLT3, phosphorylation was observed in cells expressing FLT3 mutant. To identify Lyn as one of the six types of SFK! In addition, there are no data on the binding of FLT3 mutants to Lyn, etc., suggesting that Lyn phosphate is directly caused by FLT3 mutants. They also reported that SFK inhibitors (PP1, PP2) inhibit the phosphorylation of SFK in cells expressing FLT3 mutants, which in turn inhibits cell growth. However, regarding this inhibition, it was confirmed whether the SFK inhibitors they used were Lyn or other SFKs! Furthermore, it is not clear whether SFKs other than Lyn are phosphorylated by FLT3 mutants. Therefore, it is important for the growth of cells with SFLT3 mutant phosphorylation ability of Lyn among SFK. It is unclear whether it is a therapeutic target for leukemia.

[0006] References cited in the present specification are as follows. All the contents described in these documents are cited herein as part of this specification. Any of these documents is not admitted to be prior art to this specification.

Non-patent document 1: Yokota, S., et al., (1997) 丄 eukemia, l l, 1605-9

Non-Patent Document 2: Frohling, S., et al., (2002) .Blood, 100, 4372-80

Non-Patent Document 3: Gilliland, D.G. & Griffin, J.D. (2002) .Blood, 100, 1532-42

Non-Patent Document 4: Horiike, S., et al., (1997) 丄 eukemia, 11, 1442-6

Non-Patent Document 5: Kiyoi, H., et al., (1999) .Blood, 93, 3074-80

Non-Patent Document 6: Kiyoi, H., et al., (2005). Int J Hematol, 82, 85-92

Non-Patent Document 7: Kottaridis, P.D., et al., (2001) .Blood, 98, 1752-9

Non-Patent Document 8: Nakano, Y., et al., (1999) .Br J Haematol, 104,659-64

Non-Patent Document 9: Schnittger, S., et al., (2002) .Blood, 100, 59-66

Non-Patent Document 10: Shih, L.Y., et al., (2004) 丄 eukemia, 18,466-75

Non-Patent Document 11: Shih, Y., et al, (2002) .Blood, 100, 2387-92)

Non-Patent Document 12: Hayakawa, F., et al., (2000) .Oncogene, 19,624-31

Non-Patent Document 13: Robinson, L.J., et al., (2005) .Exp Hematol, 33,469-79

Disclosure of the invention

Problems to be solved by the invention

[0007] An object of the present invention is to provide a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, and a therapeutic agent for acute myeloid leukemia.

Means for solving the problem

[0008] The present inventors have found that FLT3 / ITD and Lyn bind specifically to ITD mutations, and that Lyn is phosphorylated. And by using siRNA targeting Lyn, among the SFKs, Lyn phosphate was responsible for the abnormal activation of STAT5 in leukemia cell lines with FLT3 mutant, and the cause of abnormal growth of the same cell line. It was revealed that. Regarding SFK inhibitor PP2, it was also shown that the phosphorylation of SFK inhibited by this inhibitor is more than 80% phosphorylation of Lyn and also inhibits phosphorylation of STAT5. . [0009] That is, the present invention is a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, and whether or not the test substance inhibits the binding between the active mutant FLT3 and Lyn in vitro or in a cell. , And determining whether to inhibit Lyn phosphate in a cell expressing Z or an active mutant FLT3. Throughout this specification, the active mutant FLT3 refers to a mutant FLT3 that exhibits constitutively active FLT3 kinase activity. Typical active mutations FLT3 include FLT3 / ITD and FLT3 / TKD, both of which have FLT3 kinase activity that is constitutively active and cause abnormal signaling activity. Causes abnormal cell proliferation and causes leukemia. Although there is a difference in the mechanism of FLT3 activity between the two, no subsequent signal transduction has been reported, and in both cases, abnormal downstream activity of STAT5 is observed.

[0010] In another aspect, the present invention relates to a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, wherein the test substance is contacted with a cell expressing Lyn, and the test substance expresses Lyn expression. A method comprising determining whether to inhibit and whether Z or Lyn is capable of inhibiting STAT5 phosphate;

[0011] In another aspect, the present invention relates to an antisense oligonucleotide against Lyn gene, a ribozyme against Lyn gene, siRNA against Lyn gene, PP1, PP2, SU6656, Staurosporine, NS-187, KRX- A therapeutic agent for acute myeloid leukemia comprising a substance selected from the group consisting of 123 and BMS-354825 as an active ingredient is provided.

[0012] In still another aspect, the present invention relates to a substance that inhibits the binding between active mutant FLT3 and Lyn, a substance that inhibits phosphorylation of Lyn in cells expressing the active mutant FLT3, Provided is a therapeutic agent for acute myeloid leukemia containing as an active ingredient a substance that inhibits expression and a substance selected from the group of substances that inhibits STAT5 phosphate by Lyn.

[0013] In yet another aspect, the present invention relates to a method for treating acute myeloid leukemia, wherein the binding of active mutant FLT3 to Lyn is inhibited and Z or active mutant FLT3 is expressed. Inhibiting Lyn phosphorylation in a cell is provided.

[0014] In yet another aspect, the present invention is a method for treating acute myeloid leukemia, which inhibits Lyn expression or inhibits STAT5 phosphate by Z or Lyn A method comprising:

[0015] In still another aspect, the present invention provides a method for treating acute myeloid leukemia in which a patient in need of treatment is provided with an antisense oligonucleotide for the Lyn gene, a ribozyme for the Lyn gene, There is provided a method comprising administering a substance selected from the group consisting of siRNA, PP1, PP2, SU6656, staurosporine, NS-187, KRX-123, and BMS-354825 against a gene.

[0016] In another aspect, the present invention relates to a method for treating acute myeloid leukemia, wherein a substance that inhibits the binding of an active mutation FLT3 and Lyn, an activity to a patient in need of treatment. A substance selected from the group consisting of a substance that inhibits Lyn phosphorylation, a substance that inhibits Lyn expression, and a substance that inhibits STAT5 phosphorylation by Lyn in cells that express type FLT3. A method comprising:

Brief Description of Drawings

[0017] FIG. 1 shows phosphorylation of SFK in FLT3 / ITD-introduced 32D cells.

[Figure 2] Figure 2 shows the coupling of FLT3 and Lyn.

[Fig. 3] Fig. 3 shows phosphorylation of STAT5 and inhibition of cell proliferation by FLT3 / ITD when Lyn expression was suppressed by siRNA.

[Figure 4] Figure 4 shows the effect of SFK inhibitors on FLT3 / ITD signaling and cell proliferation.

[Fig. 5] Fig. 5 shows inhibition of proliferation of FLT3 / ITD-32D cells in C3H / HeNCj mice by PP2.

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] Non-receptor tyrosine kinase Lyn is a member of the Src family kinase (Src family kinase: SFK) and is predominantly expressed in the blood cell system. Like other SFK members, Lyn binds to the receptor and is thought to be involved in signal generation of its receptor strength. Lyn was originally discovered as a signal transduction factor for B cell antigen receptors. In recent studies, various site forces such as erythropoietin receptor, GM-CSF receptor, CSF-1 receptor, and c-kit It has been clarified that the receptor also plays a role as a transmitter of cell proliferation signals. In the present invention, as shown in the following examples, FLT3 / ITD and Lyn are specifically bound to ITD mutation in the cell, and Lyn phosphonate is associated with it. Made clear. In addition to showing in vitro that FLT3 and Lyn are directly bound, it was shown that the binding is more affinity to FLT3 / ITD than wild-type FLT3 and is dependent on FLT3 phosphate. When the FLT3 / ITD-introduced 32D cells (FLT3 / ITD-32D) were treated with either siRNA targeting Lyn or PP2 as the SFK inhibitor, the proliferation was suppressed, and Lyn and STAT5 Phosphorylation was attenuated. Furthermore, when PP2 is administered to mice transplanted with FLT3 / ITD-32D (with no treatment, they die from the tumor within 8 weeks), the onset of the tumor is prevented, the onset tumor is reduced, and the mouse survives. It was shown to be extended. These results indicate that Lyn is a very important factor for cell proliferation caused by active mutant FLT3, which is located upstream of STAT5, in addition to signal transduction specific to active mutant FLT3. It was. In addition, Lyn has been shown to be a therapeutic target for AML with the active mutation FLT3.

[0020] Screening method

In one aspect, the present invention provides a method for screening candidate substances for therapeutic agents for acute myeloid leukemia from various test substances. Screening shows whether test substance inhibits binding of active mutant FLT3 to Lyn in vitro or in cells, and inhibits Lyn phosphate in cells expressing Z or active mutant FLT3 This can be done by determining whether or not the force is strong. The ability of the test substance to inhibit the binding between active mutant FLT3 and Lyn is measured in vitro using a known binding assay using isolated active mutant FLT3 and isolated Lyn. be able to. In cells, cells expressing both the active mutations FLT3 and Lyn can be brought into contact with the test substance and assayed using known binding assays. Phosphorylation of Lyn in cells expressing active mutant FLT3 was evaluated by contacting the test substance with cells expressing active mutant FLT3 and measuring the degree of Lyn phosphorylation by a known method. can do. These assays were identified as significantly inhibiting the binding of active mutant FLT3 to Lyn, and significantly inhibiting Lyn phosphate in cells expressing Z or active mutant FLT3. These substances are considered to be candidates for inhibitors of therapeutic agents for acute myeloid leukemia. [0021] In addition, the screening method according to the present invention comprises contacting a test substance with a cell that expresses Lyn, the ability of the test substance to inhibit Lyn expression, and the STAT5 phosphate concentration by Z or Lyn. This can be done by determining whether or not it is a force that inhibits. The ability of a test substance to inhibit Lyn expression can be evaluated by measuring the amount of Lyn mRNA or the amount of protein by a known method. The ability of the test substance to inhibit STAT5 phosphate by Lyn can be assessed by measuring the degree of phosphate in the STAT5 protein using known methods. Substances identified by these assays as significantly inhibiting Lyn expression or STAT5 phosphorylation are considered candidates for inhibitors of therapeutic agents for acute myeloid leukemia.

[0022] Test substances can be obtained from various libraries such as various synthetic or natural compound libraries, combinatorial libraries, oligonucleotide libraries, peptide libraries, and the like. In addition, extracts from natural products such as bacteria, fungi, algae, plants, animals, and partially purified products may be used as test substances.

[0023] 1 ¾ early preparation

Examples of the therapeutic agent for acute myeloid leukemia of the present invention include expression (transcription of Lyn gene) such as antisense oligonucleotides, ribozymes, and molecules that cause RNA interference (RNAi) (eg, dsRNA, siRNA, shRNA, miRNA). And Z or translation) inhibitors. Such a nucleic acid can be easily designed and manufactured based on the known nucleotide sequence information of Lyn, and can bind to and inhibit the expression of the Lyn gene or mRNA encoding Lyn. General methods for controlling gene expression using antisense, ribozyme technology and RNAi technology, or gene therapy methods for expressing exogenous genes in this manner are well known in the art.

[0024] An antisense oligonucleotide refers to a nucleic acid molecule having a sequence complementary to mRNA encoding Lyn or a derivative thereof. Antisense oligonucleotides specifically bind to mRNA and inhibit protein expression by inhibiting transcription and Z or translation. Binding may be by Watson-Crick or Houdsteen-type base pair complementarity, or by triplex formation.

[0025] Ribozymes are RNA molecules with catalytic properties that can cleave target nucleic acid sequences. To express. Ribozymes generally display endonuclease, ligase or polymerase activity. Various secondary structure ribozymes are known, for example hammerhead and hairpin type ribozymes. RNA interference (RNAi) is a method of silencing target genes using double-stranded RNA molecules.

[0026] Sudden bone marrow leukemia 'therapeutic agent

In another aspect, the present invention relates to a substance that inhibits the binding between active mutant FLT3 and Lyn, a substance that inhibits phosphorylation of Lyn in a cell that expresses active mutant FLT3, and the expression of Lyn or STAT5 by Lyn. There is provided a therapeutic agent for acute myeloid leukemia comprising a substance selected from the group consisting of substances capable of inhibiting the above-mentioned phosphate as an active ingredient. Inhibitors that can inhibit Lyn include, for example, small compounds as Lyn kinase inhibitors (eg, PP1, PP2, SU6656, staurosporine, NS-187, KRX-123, and BMS-354825 (Dasachi). -B; including Dasatinib)), prediction of the binding site with the substrate based on the amino acid sequence of Lyn kinase and oligopeptide or small compound that inhibits the site, by the method of Wexler et al. (BioTechniques 39: 575-576, 2005) Kinase inhibitors that may be found, and Lyn transcription and translational repression (siRNA, antisense, etc.) can be mentioned, any of which can be used in the present invention. The therapeutic agent for acute myeloid leukemia of the present invention is useful for use in patients having the active mutation FLT3, and is considered to be particularly useful for improving prognosis.

[0027] vaginal formulation and

Although the therapeutic agent for acute myeloid leukemia of the present invention can be administered to a subject as it is, it is usually formulated and administered using a carrier used in medicine. As the carrier used in the preparation, any of those commonly used in the pharmaceutical field can be used. For example, sterile water, physiological saline, excipient, stabilizer, antioxidant, buffer, surfactant Agents and binders are preferably used. Furthermore, the therapeutic agent for acute myeloid leukemia of the present invention may be encapsulated in a microcapsule or a high molecular gel to form a sustained release preparation.

[0028] The administration route of the therapeutic agent for acute myeloid leukemia of the present invention includes oral administration, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, intracavity administration, and the like.

[0029] The dosage depends on the type of therapeutic agent for acute myeloid leukemia, the route of administration, the degree of disease, etc. Although it is appropriately selected, it is usually 0 .: g to 1000 mg / Kg, preferably: g to 10 mg / Kg.

[0030] The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2006-155281, which is the application on which the priority of the present application is based, are cited herein as part of this specification.

Example

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0032] Description

纖

The wild-type FLT3 and FLT3 / ITD (having tandem duplication of amino acids 583 to 602) genes were stably introduced into the L-3 dependent hematopoietic progenitor cell line 32D, and Wt FLT3-32D and FLT3 / It was named ITD-32D (Hayakawa, F., et al., (2000). Oncogene, 19,624-31). Cells were placed in RPMI1640 medium containing 10% urinary fetal serum (FBS) supplemented with 5ng / ml mouse IL-3 (KIRIN Brewery. Co. Japan) or FLT3 ligand (R & D Systems, Minneapolis, Minn.). And cultured.

[0033] Antibody. Reagent. Plasmid

Usagi polyclonal antibodies against Lyn, FLT3 (C-20), Jak2 (C-20), and C-Src, Fyn, Lck, c-Yes, and mouse monoclonal antibodies against Lyn are Santa Cruz Biotechnology (Santa Cruz, CA). ) Purchased from. Phosphorylation specific rabbit antibody against JAK2, STAT5, p44 / 42 MAPK, Akt, Shc, Src Fa mily (Tyr416), and non-phosphorylation specific rabbit antibody against p44 / 42 MA PK, Akt, Cell Purchased from Signaling Technology (Beverly, MA). Anti-phosphotyrosine, anti-STAT5, and anti-Hck mouse monoclonal antibodies were obtained from Transduction Laboratory (Lexington, KY). Anti-GST goat polyclonal antibody was obtained from Pharmacia (Uppsala, Sweden). SFK inhibitor was purchased from Calbiochem (La Jolla, CA) and used by dissolving in dimethyl sulfoxide (DMSO) [0034] Plasmid Lyn (foll) / pGEX, Lyn (l for expressing full-length human Lyn B or a partial fragment thereof (1-222 amino acids, 1-169 amino acids, 106-222 amino acids) as a GST fusion protein -222) / p GEX, Lyn (l-169) / pGEX, Lyn (106-222) / pGEX is LynB / pHNGAP, (Abe, A., et al "(20 04). Biochem Biophys Res Commun, 320,920 It was made by excising the Lyn gene with restriction enzymes from 6) and inserting it into the pGEX5X-l vector (Pharmacia) For the synthesis of FLT3 protein in vitro, wild-type FLT3 and FLT3 / ITD ( Plasmids were prepared by incorporating cDNA with 583-602 amino acid tandem duplications into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) and named WtFLT3 / pcDNA and FLT3 / ITD / pcDNA, respectively. FLT3 / pcDNA and 8F FLT3 / ITD / pcDNA were prepared by PCR mutagenesis using the QuikChange ™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

[0035] Preparation of cell lysate. Isolated sedimentation. Immunoblotting

To prepare the cell lysate, lxlO ⁷ cells in a cyto force-in starved state were loaded with 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, l% Nonidet P-40, 0.1% dodecyl sulfate). Sodium [SDS], lmM Sodium vanadate, ImM Fluorophenylmethylsulfol [PMSF], lmM Dithiothreitol [DTT], 10 mM sodium fluoride, and 10 g / ml aprotune, leupeptin and pepstatin Dissolved in A). Immunoprecipitation and immunoblotting were performed as previously reported (Hong, SH & Privalsky, M 丄. (2000). Mol Cell Biol, 20, 6612-25) _o

[0036] FT 3-T Am test, in vitro coupling ¾ experiment

In vitro binding experiments and protein dephosphorylation were performed with minimal improvements to previously reported methods (Hong, SH & Privalsky, M 丄. (2000). Mol Cell Biol, 20, 6612). - twenty five). Briefly, GST or various Lyn fused to GST was expressed in E. coli BL21. The manufacturer's instructions were followed for E. coli lysis, GST protein binding to Pharmacia, and purification. GST protein bound to the beads was quantified by Coomassie blue staining after SDS-PAGE. Wild-type FLT3 and FLT3 / ITD were synthesized in vitro using an in vitro translation transcription system (TnT kit, Promega, Madison, WI). The expression of various FLT3s was confirmed by immunoblotting with anti-FLT3 antibody after SDS-PAGE. 1 μg of GST protein bound to beads and 5 μl of FLT3 1% sushi serum 4 with 100 μ1 HEMG buffer (40 mM Hepes-NaOH [pH 7.8], 100 mM KCl, 5 mM MgCl 2, 10% glycerol, 0.1% Nonidet P-40, and 0.2 mM EDTA) containing rubmin (BSA) 4

2

. Incubated for 1 hour at C. The beads were washed 5 times with HEMG buffer, followed by SDS-PAGE and immunoblotting with anti-FLT3 antibody. In the FLT3 dephosphorylation reaction, the synthetic FLT3 of 5 was combined with 5 U of calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA) before the binding experiment. Incubated for 1 hour

[0037] Suppression of Lvn expression by siRNA

Two predesigned siRNAs (siRNAs targeting Lyn exon 5 or exons 7 and 8)

ID: 156196 and 156197) were purchased from Ambion (Austin, TX). BLOCK-iT Fluores cent Oligo (trademark) was purchased from Invitrogen and used as a control. A mixture of 2 Lyn siRNAs (1.5 μg each) or 3 μg of control siRNA was transiently introduced into the cells using the Nucleofector system (a maxa biosystems, Gaithersburg, MD) as recommended by the manufacturer. It was done.

[0038] PP2 cattle.

8 week old female C3H / HeNCrj mice were purchased from Charles River Japan Inc. (Atsugi, Japan). They were bred under standard conditions in accordance with the guidelines of the Nagoya University Animal Laboratory. One million FLT3 / ITD-32D cells or Wt FLT3-32D cells were administered subcutaneously on the back of mice. PP2 administration began at the time indicated in FIG. PP2 doses were determined with reference to previous reports (Nam, J.S., et al., (2002). Clin Cancer Res, 8, 2430-6). Tumor size was measured twice a week and tumor weight (TW) was calculated according to the following formula:

TW (mg) = (d ² XD) / 2

[d (mm) and D (mm) are the shortest and longest diameters of the tumor].

[0039] thigh

Introduction of FLT3 / ITD Tyrosine phosphorylation of Lvn in 32D filaments

FLT3 / ITD-32D shows IL-3 independent proliferation, and STAT5 is known to show constitutive activation in the cell (Hayakawa, F., et al., (2000). Oncogene , 19,624-31) ₀ Therefore, we focused on the pathway leading to FLT3 / ITD STAT5 and investigated phosphorylation of SFK. The results are shown in Figure 1. After culturing for 24 hours in a site force-in starved state, each cell was stimulated with IL-3 (IL3) or FLT3 ligand (FL) for 10 minutes or unstimulated (-). Cell lysates were prepared as described in the experimental method. (AXJAK2 phosphorylation. Immun blotting with anti-phosphorylated JAK2 antibody (top) or anti-JKA2 antibody (bottom) following SDS-PAGE using 10 1 lysate (IB) (B) Lyn phosphorylation.Immunoprecipitation (IP) was performed with Usagi (R) anti-Lyn antibody using 100 1 cell lysate, 90% and 10% of the immunoprecipitate, respectively. (C) Src phosphorylation (as in (B)) with IB using anti-phosphorylated mouth syn (pTyr) antibody (top photo) and mouse (M) anti-Lyn antibody (bottom photo). In addition, anti-Src antibody was used for IP and anti-phosphotyrosine antibody and anti-Src antibody were used for IB.

[0040] First, phosphorylation of JAK2, which is known as an upstream factor of STAT5, was examined using IL-3 signal, etc. However, phosphorylation of JAK2 did not occur in either wild-type FLT3 or FLT3 / ITD (Fig. 1A). Next, in order to investigate whether any SFK other than JAK2 that is known to phosphorylate STAT5 is phosphorylated, we first introduced wild-type FLT3 or FLT3 / ITD. When 32D cells (Wt FLT3-32D, FLT3 / ITD-32D) were stimulated with site force-in or were not stimulated, phosphorylation of various SFKs was examined. In FLT3 / ITD-32D, Lyn was constantly expressed. Was found. On the other hand, Wt FLT3-32D did not show Lyn phosphate after stimulation with FLT3 ligand (Fig. 1B). Src, another SFK, was also examined for its phosphoric acid state, but there was no difference between the two cells regardless of the presence or absence of site force in (Fig. 1C). This suggests that Lyn force is an upstream factor of STAT5 in FLT3 / ITD signal transduction and that the signal transduction is involved in the cell proliferation effect resulting from FLT3 / ITD force. The experiment shown in Fig. 1B revealed that the phosphorylation of SFK, reported by Robinson et al., Is abnormally activated by FLT3 / ITD, is the phosphorylation of Lyn. In this report, Lyn phosphate was reported by wild-type FLT3, but this fact was not observed in our experiment.

[0041] The binding of Lvn and FLT3 is clearly strong in FLT3 / ITD and depends on the phosphorylation of FLT3.

Next, to investigate whether the binding of FLT3 and Lyn is specific to FLT3 / ITD, anti-FLT3 A coprecipitation experiment was performed in which the antibody was immunoprecipitated and immunoblotted with an anti-Lyn antibody. The result is shown in figure 2. After culturing for 6 hours in a cyto force-in starved state, the cells were stimulated with a site force in as described in Fig. 1, and the cell lysate was prepared. (A) Binding of FLT3 and Lyn in the cell. 100 1 lysate was used for immunoprecipitation with anti-FLT3 antibody. 90% and 10% of the immunoprecipitates were subjected to SDS-PAGE and then immunoblotted with anti-Lyn antibody (M) (top) or anti-FLT3 antibody (bottom). (B) A specular coprecipitation experiment using anti-Lyn antibody (R) for immunoprecipitation and anti-FLT3 antibody or anti-Lyn antibody (M) for immunoblotting was performed in the same manner as (A). It was. (OFLT3 vs. Lyn in vitro binding experiment. Wild type and mutant FLT3 were synthesized in vitro, and their expression was confirmed to be equivalent by imknob fitting with anti-FLT3 antibody (data not shown)). Amount (5 μl) of FLT3 protein was precipitated with 1 μg of GST (G) or Dartathione beads conjugated with Lyn (L) fused to GST. The same FLT3 protein used in the binding experiment was immunoprecipitated with anti-FLT3 antibody, and after SDS-PAGE, it was immunoprecipitated with anti-phosphotyrosine antibody or anti-FLT3 antibody. Muno blotted (right photo) (D) In vitro binding experiment after dephosphorylation, FLT3 synthesized in vitro was shown in the figure before the binding experiment as shown in the calf With or without dephosphorylate reaction with or without intestinal alkaline phosphatase (CIP) In vitro binding experiments and confirmation of the phosphorylation state of FLT3 were performed as described in (C) (E) Schematics of various Lyn and FLT3 constructs FLT3 / ITD 4FWt FLT3 and 8F FLT3 / black circles are the same as the amino acid sequences of Wt FLT3 and FLT3 / ITD, respectively (F) The binding region of Lyn Identification GST fusion proteins of various parts of Lyn were used as shown and binding experiments were performed as described in (C) 90% of the precipitate was immunoblotted with anti-FLT3 antibody 10% were immunoblotted with anti-GST antibody (G) Identification of FLT3 binding sites In vitro binding experiments and confirmation of phosphorylation status were performed as described in (C).

No binding (coprecipitation) between Lyn and FLT3 was observed in FLT3 / ITD-32D with Wt FLT3-32D, even when stimulated with FLT3 ligand (Figure 2A). This mutation-specific binding of FLT3 and Lyn is specular using anti-Lyn antibody for immunoprecipitation and anti-FLT3 antibody for immunoblotting. This was also confirmed in the experimental coprecipitation experiment (Fig. 2B). Furthermore, in order to investigate whether the coupling is direct or via other factors, a coupling experiment was conducted in a test tube. It was suggested that the N-terminal half containing Lyn SH3 and SH2 regions and the GST fusion protein were bound to synthetic FLT3 in vitro, and both were bound directly (Fig. 2C left panel). Of note, wild-type FLT3 is also weaker than FLT3 / ITD, but shows binding to Lyn, and the mutation specificity in the binding between FLT3 and Lyn is considered to be not absolute. In many cases, the binding of receptor tyrosin kinase (RTK) and SFK depends on tyrosine phosphorylation of the receptor! /, (Boggon, TJ & Eck, MJ (2004). Oncogene, 23, 7918-27. ). Therefore, we investigated the phosphorylation state of synthetic FLT3 used in the in vitro binding experiments, and examined whether this affects the binding of FLT3 and Lyn in this system. FLT3 synthesized by the system using the rabbit reticulocyte lysate caused self-tyrosine phosphorylation, and the phosphorylation of FLT3 / ITD was stronger than that of wild-type FLT3 (Fig. 2C, right panel). . This suggests that the binding of FLT3 and Lyn depends on the tyrosine phosphorylation of FLT3, and the difference in the degree of phosphorylation of FLT3 may cause the difference in binding! /.

In order to investigate the dependence of this binding on phosphate, an in vitro binding experiment was performed using dephosphorylated FLT3. As expected, dephosphorylation of FLT3 lost its binding to Lyn in both wild-type and mutant FLT3, suggesting its dependence on FLT3 phosphate. In many cases, the SH2 region is involved in the recognition of phosphate synthase. Therefore, to further clarify the dependence of this binding on FLT 3 tyrosine phosphate, Lyn's SH2 region force Involved, I investigated the power of the ru. Furthermore, the effect of the tyrosine ferrolanine substitution on the parasial region, which is considered to be the most probable Lyn binding region of FLT3, was examined. In vitro binding experiments using fusion proteins of various parts of Lyn and GST as shown in Fig. 2E and synthetic FLT3 / ITD revealed the involvement of the Lyn SH2 region in this binding ( (Figure 2F). The FLT3 / ITD used has a tandem duplication of the portion corresponding to the 583-602 amino acids of wild-type FLT3 in the paramembrane region. For wild-type FLT3 and FLT3 / ITD, the 4 and 8 tyrosines present in this region were replaced with ferrolanins (named 4F and 8F substitutions, respectively). They were named Wt FLT3 and 8 F FLT3 / ITD (Figure 2E). 4F Wt FLT3 and 8F F in in vitro coupling experiments LT3 / ITD showed reduced binding to Lyn (approximately 30% of Wt FLT3 and FLT3 / ITD, respectively) (Figure 2G left panel). In addition, the substitution of 4F and 8F markedly decreased the tyrosin phosphate of wild-type FLT3 and FLT3 / ITD (Fig. 2G right panel). These data suggest that the substituted thiocin is a phosphorylation site in wild-type FLT3 and FLT3 / ITD autophosphorylation, and that tyrosine phosphorylation is recognized in the SH2 region of Lyn. . The binding of wild-type and mutant FLT3 and Lyn shown in Fig. 2A-G, the identification of the binding site, and the dependence of the binding on FLT3 phosphate were all clarified for the first time in the present invention. The

Inhibition of specific Lvn signal by siRNA specifically inhibits cell multiplication by FLT3 / ITD

We investigated the significance of Lyn activity in FLT3 / ITD site-independent growth by suppressing Lyn expression with siRNA designed based on Lyn-specific DNA sequences. The results are shown in Figure 3. Suppression of Lyn expression by siRNA. Millions of FLT3 / ITD-32D cultured in the presence of IL_3 were introduced into the cells using a Nucleofector system with 3 μg of control siRNA (C) or 1.5 μg of each mixture of two L yn siRNAs. It was. Twenty-four hours after introduction, the cells were washed and placed in a site force-in starvation state for 24 hours. Cell lysates were then prepared, and after SDS-PAGE, immunoblotting was performed with antibodies against Lyn, c-Src, Fyn, Lck, c-Yes and Hck as shown. (B) Specific inhibition of STAT5. The same lysate as in (A) was used for immunoblotting with phosphate-specific antibodies as shown. The expression of each signaling factor other than SFK was confirmed by immunoblotting with the corresponding non-specific antibody (data not shown). (C), (D) Effect of suppression of Lyn expression by siRNA on the proliferation of various 32D cells introduced with FLT3. Wt FLT3-32 D and FLT3 / ITD-32D were cultured in the presence of IL-3 and introduced with siRNA as described in (A). Cells were washed 24 hours after introduction, and 5 ng / ml IL-3 (IL3), FLT3 ligand (FL), or no cytodynamic force-in (-) culture medium as shown, 2 X 10 ⁵ cells / ml. Resuspended at a density of (Time 0). The number of viable cells at each time point was measured by the trypan blue exclusion method, and a graph was created by calculating the ratio to the value at time 0 (2 X 10 ⁵ cells / ml). Each experiment was performed twice, and the mean and standard deviation are shown. [0045] In FLT3 / ITD-32D, it was confirmed by force immunoblotting that Lyn siRNA suppressed Lyn expression by approximately 90%. Furthermore, the specificity of expression suppression was confirmed by immunoblotting. That is, the expression of all five SFKs other than Lyn was clearly affected! / No (Fig. 3A). The effect of Lyn expression suppression on FLT3 / ITD signaling was examined by immunoblotting with phosphate-specific antibodies against each signaling factor. The SFK phosphate was markedly reduced to about 20%. The expression of SFKs other than Lyn is affected, and taking into account their strength, this decrease in SFK phosphate is thought to be caused by suppression of Lyn expression. This means that in FLT3 / ITD-32D cells, the phosphate detected by anti-phosphate SFK antibody is mainly Lyn phosphate, and this antibody is FLT3 / ITD-32D. It can be used to detect Lyn phosphate in cells. STAT5 phosphorylation was reduced to about 50% of the control, but phosphorylation of MAP kinase, Akt, and She was not affected (Fig. 3B). As shown here, the phosphate phosphate recognized by the anti-phosphate SFK antibody was shown for the first time only by showing that Lyn-specific expression suppression significantly decreased (over 80%). (80% or more) can be expressed as phosphorylation of Lyn. In the previous report of Robinson et al., Such experiments were carried out! /, Na! / ヽ, so it is unclear which SFK phosphate was observed.

[0046] The specific damage of STAT5 phosphorylation by the system is indicated by the fact that Lvn is the STAT5 fluency at the end of signal transmission.

The effect of Lyn siRNA on the proliferation of FLT3-introduced 32D was examined. In the absence of IL-3, the growth of FLT3 / ITD-32D was markedly suppressed by Lyn siRNA (Figure 3D). On the other hand, in the presence of IL-3, the proliferation of Wt FLT3-32D cells and FLT3 / ITD-32D cells was not affected. Proliferation of Wt FLT3-32D cells with FLT3 ligand was also unaffected (Figure 3C and 3D). These results suggest that Lyn is more important in cell proliferation induced by FLT3 / ITD signals than cell proliferation induced by wild-type FLT3 and IL-3 signals. In addition, SFK inhibitors such as PP1 and PP2 used in the above-mentioned report by Robinson et al. Are not clear in SFK. Therefore, SFK inhibitors suppress cell proliferation. However, it is not possible to identify which SFK is the inhibition, that is, which SFK is important for cell proliferation. Shown in the present invention Thus, Lyn is important for the first time by suppressing Lyn-specific expression. That is, this is the first time that Lyn has been identified as important for cell growth in cells expressing FLT3 mutants.

[0047] Inhibition of FLT3 / ITD- 32D fine cell proliferation by SFK inhibitor PP2

To further confirm the importance of Lyn in cell proliferation by FLT3 / ITD, the SFK inhibitor PP2 (Hanke, JH, et al., (1996). J Biol Chem, 271, 695-701), Wt FLT3-32D And the effect of FLT3 / ITD-32D on proliferation. Both cell lines were cultured for 24 hours with various concentrations of PP2. The results are shown in Fig. 4. (A) Concentration-dependent growth inhibition by PP2. Wt FLT3-32D (Wt) or FLT3 / ITD-32D (Mut) was cultured in the presence of IL-3 in the absence of Z for 24 hours with the addition of PP2 at the indicated concentration. The number of viable cells at each concentration after 24 hours was counted by the trypan blue exclusion method. A graph was created by calculating the ratio of each value to the value at 0 M in%. Each experiment was performed twice in triplicate, and the mean and standard deviation are shown. (B) Proliferation curve of cells treated with PP2. FLT3 / ITD-32D was cultured with IL-3 (2 ng / ml) and ΡΡ2 (10 μΜ) supplemented with the combination shown. Viable cell count and graph creation are the same as in Figure 3C. Each experiment was performed twice in triplicate, and the mean and standard deviation are shown. (C), (D) Signal inhibition in PP2-treated FLT3 / ITD-32D. 10 / z M PP2 was added to FLT3 / ITD-32D cultured in the absence of Z in the presence of IL-3. At the time indicated, the cell lysate was prepared as described above. FLT3 phosphate was examined in the same manner as in Figure 1B, except that anti-FLT3 antibody was used for immunoprecipitation. Other signaling factors, phosphate, were examined as in Figure 3B. The expression of each factor or the efficiency of FLT3 immunoprecipitation between each time point was confirmed by immunoblotting using the corresponding non-specific antibody.

[0048] The growth inhibitory effect was clearly concentration-dependent and was strong in FLT3 / ITD-32D cultured in the absence of IL-3. GI for FLT3 / ITD-32D in the absence of IL-3 is the presence of IL-3

50

The lower Wt FLT3-32D and FLT3 / ITD-32D were about one third (5 Μ vs. 17.7 μ.) (Fig. 4A). The cell growth curves of FLT3 / ITD-32D in the presence of IL-2 in the presence or absence of PP2 at 10 μΜ were also compared. In the absence of IL-3, FLT3 / ITD-32D proliferation was completely inhibited by ΡΡ2. However, some suppression of proliferation was observed even in the presence of IL-3 (Fig. 4). Ρ Inhibition of cell growth by P2 is considered to be more effective against that induced by FLT3 / ITD than cell proliferation induced by IL-3, which suggests that P P2 is effective against cancers caused by FLT3 / ITD. Has the potential to work as an anti-tumor agent. Next, the effect of PP2 on the signaling factor induced by FLT3 / ITD or IL-3 was observed over time using anti-phosphorylated antibodies. In FLT3 / ITD-32D, the phosphorylation state of FLT3 was not changed by treatment with 10 μΜ of ΡΡ2 (Fig. 4C, upper panel). However, in the absence of IL-3, the phosphorylation of SFK, which mainly consists of Lyn phosphorylation, disappeared rapidly within 1 hour, and the phosphorylation of STAT5 also disappeared within 6 hours. Mild attenuation of MAP kinase phosphate was observed after 12 hours, and phosphorylation of Akt and She was not affected (Fig. 4C). JAK2 phosphorylation was not observed at any time (data not shown). On the other hand, in the presence of IL-3, JAK2 phosphate was observed, which was slightly suppressed after 12 hours. In line with the fact that JAK2 phosphate remained, phosphorylation of STAT5 also remained. Attenuation of MAP kinase phosphorylation was observed to the same extent as in the absence of IL-3 (Figure 4D). Akt and She phosphates were unaffected (data not shown). From these results, it is determined whether the phosphorylation inhibition of signaling factors is the direct effect of Lyn inhibition, the force of SFK inhibition other than Lyn, or the inhibition of kinases other than SFK. I couldn't do it. However, signal inhibition by PP2 appears to occur more strongly with respect to S TAT5, confirming the hypothesis that it is an upstream factor of Lyn force TAT5.

Therapeutic effect of PP2 on needle injury caused by FT / H / TTD in a mouse model

In order to investigate the possibility of an SFK inhibitor as a therapeutic agent for cancer caused by FLT3 / ITD, a mouse model of FLT3 / ITD developed previously (FLT3 / ITD-32 D was not irradiated). Made by inoculating mice) (Zhao et al., (20 00) 丄 eukemia, 14,374-8.). The results are shown in FIG. Six mice were inoculated subcutaneously with FLT3 / ITD-3 2D on the first day, 5 μg or 10 μg ΡΡ2 or DMSO alone dissolved in DMSO twice a week, day 4 force 63 days It was administered intraperitoneally to the eyes. FLT3 / ITD-32D (1 × 10 ⁶ cells) was inoculated subcutaneously into 8 week old female C3H / HeNCj mice. (A) Prior administration of PP2 prevents tumor development. Each mouse received 5 g or 10 g of PP2 or DMSO alone in 100 1 DMSO twice a week, ip from day 4 to day 63. Co Troll mice were treated with DMSO and died at week 8 (shown in cross). (B) Administration of PP2 after tumor onset suppresses tumor progression.

All mice inoculated with FLT3 / ITD-32D developed tumors by day 25. From Day 2 to Day 63, until the 63rd day, the indicated amount of PP2 dissolved in 100 1 DMSO was administered intraperitoneally twice a week.

[0050] In the control mice, the subcutaneous tumor rapidly increased from the 25th day, and all mice died by the 45th day. In contrast, the group that received 10 g of PP2 did not develop obvious tumors. In the group receiving 5 / z g of PP2, tumors developed from day 25, but their growth was markedly suppressed over the entire observation period. The tumor weight was approximately one-third that of tumors in the control group (Figure 5A).

[0051] The effect of PP2 on tumor progression was next examined. Six mice were inoculated with FLT3 / ITD-32D and all developed tumors by day 25. These mice were given PP2 or DMSO after the tumor developed (day 25). In control mice, subcutaneous tumors grew rapidly and all mice died by day 45. On the other hand, in the group given 30 g of PP2, tumor progression was slow and all mice were still alive at day 63. Furthermore, in the group administered with 60 g of PP2, stronger growth inhibition was observed. That is, by day 35, the tumor had shrunk until it could not be detected (Fig. 5B). Both mice in this group remained alive without tumor growth for more than 20 weeks after the end of PP2. These results indicate that PP2 is a safe and effective drug for the prevention and treatment of tumors with FLT3 / ITD in the amounts used here.

Industrial applicability

[0052] The present invention is useful for screening candidate substances for therapeutic agents for acute myeloid leukemia and for treating acute myeloid leukemia.

Claims

The scope of the claims

[1] A method for identifying candidate substances for the treatment of acute myeloid leukemia, in which the active mutant FLT3 is contacted with Lyn in the presence of the test substance, and the test substance binds to the active mutant FLT3 and Lyn. And determining whether or not it inhibits phosphorylation of Lyn in cells expressing Z or active mutant FLT3.

[2] A method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, comprising contacting a test substance with a cell that expresses Lyn, whether the test substance inhibits Lyn expression, and whether Z or Lyn Determining whether to inhibit STAT5 phosphorylation.

[3] An antisense oligonucleotide against the Lyn gene, a ribozyme against the Lyn gene, siRNA against the Lyn gene, PP1, PP2, SU6656, staurosporine, NS-187, KRX-123, and BMS-354825 A therapeutic agent for acute myeloid leukemia containing the active substance as an active ingredient.

[4] Substances that inhibit the binding of active mutant FLT3 and Lyn, substances that inhibit Lyn phosphate in cells that express active mutant FLT3, substances that inhibit Lyn expression, and S by Lyn A therapeutic agent for acute myeloid leukemia comprising as an active ingredient a substance selected from the group consisting of substances that inhibit phosphorylation of TAT5.

[5] A method for treating acute myeloid leukemia, which inhibits the binding of active mutant FLT3 to Lyn, and inhibits Lyn phosphate in cells expressing Z or active mutant FLT3. A method involving harming.

[6] A method of treating acute myeloid leukemia, comprising inhibiting Lyn expression and inhibiting STAT5 phosphate by Z or Lyn.

[7] A method for treating acute myeloid leukemia, in which patients who need treatment have antisense oligonucleotides against the Lyn gene, ribozymes against the Lyn gene, siRNA against the Lyn gene, PP1, PP2, SU6656, Administering a substance selected from the group consisting of taurosporine, NS-187, KRX-123, and BMS-354825.

[8] A method for treating acute myeloid leukemia, in which a patient in need of treatment is treated with a substance that inhibits the binding between active mutant FLT3 and Lyn, and the expression of Lyn in cells expressing active mutant FLT3. Substances that inhibit phosphorylation, substances that inhibit Lyn expression, and STAT5 phosphorylation by Lyn Administering a substance selected from the group consisting of substances that inhibit oxidation.