JPWO2007142139A1 - Method for identifying candidate substances for therapeutic agents for acute myeloid leukemia - Google Patents

Abstract

Contact the active mutant FLT3 with Lyn in the presence of the test substance, whether the test substance inhibits the binding of the active mutant FLT3 and Lyn, and / or Lyn in the cell expressing the active mutant FLT3 Disclosed is a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia by determining whether or not phosphorylation is inhibited. In addition, by contacting a test substance with a cell expressing Lyn and determining whether the test substance inhibits Lyn expression and / or inhibits phosphorylation of STAT5 by Lyn, A method for identifying a candidate substance for a therapeutic agent for myeloid leukemia is also disclosed. Substances identified by this method are useful as active ingredients in therapeutic agents for acute myeloid leukemia.

Description

  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.

  Fms-like tyrosine kinase 3 (FLT3) is a receptor belonging to the type III receptor tyrosine kinase together with KIT, FMS, PDGF receptor 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 hemocyte progenitor cells. When the ligand binds to the receptor, the receptor forms a dimer through the ligand, whereby the receptor is autophosphorylated and the downstream signaling factor is activated. Signals by FLT3 play a very important role in hematopoiesis by controlling stem cell differentiation and proliferation.

  Recently, active mutation FLT3 was found frequently in adult acute myeloid leukemia (AML) cases. The presence of the active mutation FLT3 constitutively activates the receptor and its downstream signaling factor and promotes cell growth. As the active mutation FLT3, the FLT3 paramembrane region (JMD) gene has an internal tandem duplication (ITD) type (FLT3 / ITD), and the tyrosine kinase domain has a point mutation (FLT3). / TKD) (Yamamoto Y, et al., (2001). Blood, 97, 2434-9) is known. FLT3 / ITD mutations have been reported in 20% of AML cases (Yokota, S., et al., (1997). Leukemia, 11, 1605-9). A number of different variations are known (Kiyoi H., et al., (1998). Leukemia, 12, 1333-7; Hayakawa F., et al., (2000). Oncogene, 19, 624-31.). It has been shown in animal models that abnormal signaling caused by the active mutation FLT3 is involved in the onset and progression of leukemia as a first hit or second hit. The presence of active mutant FLT3 is a poor prognostic factor for AML and has been reported to be associated with progression of AML and myelodysplastic syndrome (relapse, leukemia) (Frohling, S., et al. , (2002) .Blood, 100,4372-80; Gilliland, DG & Griffin, JD (2002) .Blood, 100,1532-42; Horiike, S., et al., (1997) .Leukemia, 11,1442 -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) .Blood, 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). Leukemia, 18, 466-75; Shih, LY, et al, (2002). Blood, 100, 2387-92). Therefore, abnormal FLT3 signaling is thought to play an important role in the onset and progression of leukemia.

  The present inventors first introduced FLT3 / ITD into 32D and BA / F3, which are cell lines showing IL-3-dependent growth, and FLT3 / ITD in these cases is IL-3-independent growth and constitutive. Has been shown to cause activation of STAT5, a novel MAP kinase (Hayakawa, F., et al., (2000). Oncogene, 19, 624-31). In particular, activation of STAT5 is a phenomenon that is not observed in cells transfected with wild-type FLT3, which is strongly suggested to be the cause of IL-3-independent growth, but mutant FLT3 and STAT5 There is no known signal transduction pathway.

  Robinson et al. (Robinson, LJ, et al., (2005). Exp Hematol, 33, 469-79) phosphorylate Lyn, a member of Src family kinases (SFK), upon stimulation with FLT3 ligand in cells expressing wild-type FLT3. To be reported. It also shows abnormal phosphorylation of SFK in cell lines expressing FLT3 mutants, but due to the difference in the experimental system from that used for wild-type FLT3, it is phosphorylated in cells expressing FLT3 mutants. , It is not enough to identify Lyn among 6 types of SFK. Moreover, no data such as binding of FLT3 mutant to Lyn are shown, and it does not suggest that phosphorylation of Lyn is directly caused by FLT3 mutant. They also reported that SFK inhibitors (PP1, PP2) inhibit the phosphorylation of SFK in cells expressing FLT3 mutants, which in turn inhibits cell proliferation. However, regarding this inhibition, it has not been 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 was unclear whether Lyn phosphorylation in SFK is important for the growth of cells with FLT3 mutant, that is, it could be a therapeutic target for leukemia.

References cited in this specification are as follows. All the contents described in these documents are cited here as a part of this specification. None of these documents is admitted to be prior art to this specification.
Yokota, S., et al., (1997) .Leukemia, 11, 1605-9 Frohling, S., et al., (2002) .Blood, 100,4372-80 Gilliland, DG & Griffin, JD (2002) .Blood, 100,1532-42 Horiike, S., et al., (1997) .Leukemia, 11, 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) .Blood, 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) .Leukemia, 18,466-75 Shih, LY, et al, (2002) .Blood, 100,2387-92) Hayakawa, F., et al., (2000) .Oncogene, 19,624-31 Robinson, LJ, et al., (2005) .Exp Hematol, 33, 469-79

  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.

  The present inventors have found that FLT3 / ITD and Lyn bind specifically to the ITD mutation, and that Lyn is phosphorylated accordingly. And by using siRNA targeting Lyn, phosphorylation of Lyn among SFKs is responsible for the abnormal activation of STAT5 in leukemia cell lines with FLT3 mutants and the abnormal growth of the same cell lines It revealed that. In addition, the SFK inhibitor PP2 showed that phosphorylation of SFK inhibited by this inhibitor was more than 80% phosphorylation of Lyn and also inhibited phosphorylation of STAT5.

  That is, the present invention is a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, wherein the test substance inhibits the binding between the active mutant FLT3 and Lyn in vitro or in a cell, and / or Alternatively, a method comprising determining whether or not Lyn phosphorylation is inhibited in a cell expressing an active mutant FLT3 is provided. As used herein, the active mutant FLT3 refers to a mutant FLT3 that exhibits a constitutively activated FLT3 kinase activity. Representative active mutations FLT3 include FLT3 / ITD and FLT3 / TKD, both of which have constitutively activated FLT3 kinase activity, abnormally activated signaling, and abnormal cell proliferation Cause leukemia. Although both differ in the mechanism of FLT3 activation, no subsequent signal transduction has been reported, and in both cases, abnormal activation of downstream STAT5 is observed.

  In another aspect, the present invention provides a method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, wherein a test substance is contacted with a cell expressing Lyn, and whether or not the test substance inhibits Lyn expression. And / or determining whether to inhibit phosphorylation of STAT5 by Lyn.

  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-123, and BMS. A therapeutic agent for acute myeloid leukemia containing a substance selected from the group consisting of -354825 as an active ingredient is provided.

  In yet another aspect, the present invention inhibits the expression of Lyn, a substance that inhibits the binding between active mutant FLT3 and Lyn, a substance that inhibits phosphorylation of Lyn in cells that express active mutant FLT3 Provided is a therapeutic agent for acute myeloid leukemia comprising as an active ingredient a substance selected from the group consisting of a substance and a substance that inhibits phosphorylation of STAT5 by Lyn.

  In yet another aspect, the present invention relates to a method for treating acute myeloid leukemia, wherein the method inhibits the binding between active mutant FLT3 and Lyn and / or in a cell expressing active mutant FLT3. A method comprising inhibiting phosphorylation of Lyn is provided.

  In yet another aspect, the present invention provides a method of treating acute myeloid leukemia comprising inhibiting Lyn expression and / or inhibiting STAT5 phosphorylation by Lyn .

  In yet another aspect, the present invention relates to a method for treating acute myeloid leukemia, in which an antisense oligonucleotide for Lyn gene, a ribozyme for Lyn gene, siRNA for Lyn gene, A method is provided comprising administering a substance selected from the group consisting of PP1, PP2, SU6656, staurosporine, NS-187, KRX-123, and BMS-354825.

  In still another aspect, the present invention provides a method for treating acute myeloid leukemia, wherein a substance that inhibits binding between active mutant FLT3 and Lyn, active mutant FLT3, is administered to a patient in need of treatment. Providing a method comprising administering a substance selected from the group consisting of a substance that inhibits phosphorylation of Lyn, a substance that inhibits Lyn expression, and a substance that inhibits phosphorylation of STAT5 by Lyn To do.

FIG. 1 shows phosphorylation of SFK in FLD3 / ITD-introduced 32D cells. FIG. 2 shows the combination of FLT3 and Lyn. FIG. 3 shows phosphorylation of STAT5 and inhibition of cell proliferation by FLT3 / ITD when Lyn expression is suppressed by siRNA. FIG. 4 shows the effect of SFK inhibitors on FLT3 / ITD signaling and the resulting cell proliferation. FIG. 5 shows suppression of proliferation of FLT3 / ITD-32D cells in C3H / HeNCj mice by PP2.

  The non-receptor tyrosine kinase Lyn is a member of the 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 from that receptor. Lyn was originally discovered as a B cell antigen receptor signaling factor, but recent research has shown that cell proliferation of various cytokine receptors such as erythropoietin receptor, GM-CSF receptor, CSF-1 receptor, and c-kit. It was also revealed that it has a role as a signal transduction factor.

  In the present invention, as shown in the Examples below, it was clarified that FLT3 / ITD and Lyn bind specifically to the ITD mutation in the cell, and that Lyn is phosphorylated accordingly. In addition, we showed that FLT3 and Lyn were directly bound in vitro, and that the binding was more affinity to FLT3 / ITD than wild-type FLT3 and was dependent on FLT3 phosphorylation. When the FLT3 / ITD-introduced 32D cells (FLT3 / ITD-32D) were treated with either LRNA-targeted siRNA or the SFK inhibitor PP2, 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, the tumor will die 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 located upstream of STAT5 in signal transduction specific to the active mutant FLT3 and is a very important factor for cell proliferation caused by the active mutant FLT3. Furthermore, Lyn has been shown to be a therapeutic target for AML with the active mutation FLT3.

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 determines whether the test substance inhibits the binding of active mutant FLT3 and Lyn in vitro or in cells and / or whether phosphorylation of Lyn is inhibited in cells expressing active mutant FLT3 This can be done by determining. 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 by a known binding assay. Lyn phosphorylation in cells expressing active mutant FLT3 is assessed by contacting cells expressing active mutant FLT3 with the test substance and measuring the degree of phosphorylation of Lyn by known methods be able to. Substances identified by these assays as significantly inhibiting binding of active mutant FLT3 to Lyn and / or significantly inhibiting phosphorylation of Lyn in cells expressing active mutant FLT3 Is considered to be a candidate for a suppressant for the treatment of acute myeloid leukemia.

  Further, the screening method according to the present invention comprises contacting a test substance with a cell that expresses Lyn, and whether the test substance inhibits Lyn expression and / or inhibits STAT5 phosphorylation by Lyn. This can be done by determining. 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 a test substance to inhibit phosphorylation of STAT5 by Lyn can be evaluated by measuring the degree of phosphorylation of STAT5 protein by a known method. 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.

  The test substance can be obtained from libraries such as various synthetic or natural compound libraries, combinatorial libraries, oligonucleotide libraries, peptide libraries and the like. Moreover, you may use the extract from natural products, such as bacteria, fungi, algae, a plant, and an animal, or its partially purified product as a test substance.

Lyn gene expression inhibitor Examples of the therapeutic agent for acute myeloid leukemia of the present invention include antisense oligonucleotides, ribozymes, molecules that cause RNA interference (RNAi) (for example, dsRNA, siRNA, shRNA, miRNA), Lyn, etc. Examples thereof include inhibitors of gene expression (transcription and / or translation). 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.

  An antisense oligonucleotide represents 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 / or translation. Binding may be by Watson-Crick or Hoogsteen-type base pair complementarity, or by triplex formation.

  A ribozyme represents an RNA molecule having catalytic properties that can cleave a target nucleic acid sequence. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Various ribozymes of secondary structure, such as hammerhead type and hairpin type ribozymes are known. RNA interference (RNAi) refers to a technique of silencing a target gene using a double-stranded RNA molecule.

In another aspect of the therapeutic agent for acute myeloid leukemia , 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 that express active mutant FLT3, and Lyn A therapeutic agent for acute myeloid leukemia comprising as an active ingredient a substance selected from the group consisting of substances that inhibit the expression of STAT5 or phosphorylation of STAT5 by Lyn is provided. 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 (dasatinib) ; Including Dasatinib)), prediction of the binding site to the substrate based on the amino acid sequence of Lyn kinase, and oligopeptides or small compounds that inhibit the site, as found in the method of Wexler et al. (BioTechniques 39: 575-576, 2005) These may include kinase inhibitors, and suppression of Lyn transcription / translation (siRNA, antisense, etc.), 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.

Pharmaceutical Formulation and Administration The therapeutic agent for acute myeloid leukemia of the present invention can be administered as it is to a subject, but 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, excipients, stabilizers, antioxidants, buffers, surfactants, A binder or the like is preferably used. Furthermore, the therapeutic agent for acute myeloid leukemia of the present invention may be encapsulated in a microcapsule or a polymer gel to form a sustained release preparation.

  Examples of the administration route of the therapeutic agent for acute myeloid leukemia of the present invention include oral administration, intravenous administration, intradermal administration, subcutaneous administration, intramuscular administration, intracavitary administration and the like.

  The dose is appropriately selected according to the type of acute myeloid leukemia therapeutic agent, the route of administration, the degree of disease, etc., but is usually 0.1 μg to 1000 mg / Kg, preferably 1 μg to 10 mg / Kg.

  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.

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

Method
cell
The wild type FLT3 and FLT3 / ITD (with 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 are cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS) supplemented with 5ng / ml mouse IL-3 (KIRIN Brewery. Co., Japan) or FLT3 ligand (R & D Systems, Minneapolis, Minn.). It was.

Antibodies, reagents, plasmids
Rabbit polyclonal antibodies against Lyn, FLT3 (C-20), Jak2 (C-20), and c-Src, Fyn, Lck, c-Yes, and mouse monoclonal antibodies against Lyn are available from Santa Cruz Biotechnology (Santa Cruz, CA) We purchased more. Phosphorylation specific rabbit polyclonal antibody against JAK2, STAT5, p44 / 42 MAPK, Akt, Shc, Src Family (Tyr416), and non-phosphorylated rabbit polyclonal antibody against p44 / 42 MAPK, Akt, Cell Signaling Technology (Beverly , MA). Anti-phosphotyrosine, anti-STAT5, and anti-Hck mouse monoclonal antibodies were obtained from the Transduction Laboratory (Lexington, KY). Anti-GST goat polyclonal antibody was obtained from Pharmacia (Uppsala, Sweden). SFK inhibitor was purchased from Calbiochem (La Jolla, Calif.) And dissolved in dimethyl sulfoxide (DMSO).

  Plasmids Lyn (full) / pGEX and Lyn (1-222) / pGEX for expressing human full-length Lyn B or partial fragments thereof (1-222 amino acids, 1-169 amino acids, 106-222 amino acids) as GST fusion proteins , Lyn (1-169) / pGEX and Lyn (106-222) / pGEX are restriction enzymes from LynB / pHNGAP, (Abe, A., et al., (2004). Biochem Biophys Res Commun, 320, 920-6). It was made by excising the Lyn gene and inserting it into the pGEX5X-1 vector (Pharmacia). For the synthesis of FLT3 protein in vitro, a plasmid was constructed in which cDNAs of wild-type FLT3 and FLT3 / ITD (having tandem duplication of 583-602 amino acids) were incorporated into pcDNA3.1 vector (Invitrogen, Carlsbad, CA) They were named WtFLT3 / pcDNA and FLT3 / ITD / pcDNA, respectively. 4F Wt FLT3 / pcDNA and 8F FLT3 / ITD / pcDNA were generated by PCR mutagenesis using QuikChange ™ site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

Preparation of cell lysate, immunoprecipitation, immunoblotting To prepare cell lysate, 1 x 10 ⁷ cells in a starved state of cytokine were added to 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT], 10 mM sodium fluoride, and 10 μg / ml aprotinin, leupeptin and Dissolved in pepstatin A). Immunoprecipitation and immunoblotting were performed as previously reported (Hong, SH & Privalsky, ML (2000). Mol Cell Biol, 20, 6612-25).

FLT3-Lyn in vitro binding experiments In vitro binding experiments and protein dephosphorylation were performed with minimal modifications to previously reported methods (Hong, SH & Privalsky, ML (2000). Mol Cell Biol, 20,6612-25).
Briefly, GST or various Lyn fused to GST was expressed in E. coli BL21. Escherichia coli lysis, binding of GST protein to glutathione beads (Pharmacia), and purification were according to the manufacturer's instructions. The 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 the in vitro translation transcription system (TnT kit, Promega, Madison, WI). The expression of various FLT3 was confirmed by immunoblotting with anti-FLT3 antibody after SDS-PAGE. 1 μg of GST protein and 5 μl of FLT3 bound to beads were added to 100 μl of HEMG buffer (40 mM Hepes-NaOH [pH 7.8], 100 mM KCl, 5 mM MgCl ₂ , 10% glycerol containing 1% bovine serum albumin (BSA). , 0.1% Nonidet P-40, and 0.2 mM EDTA) for 1 hour at 4 ° C. The beads were washed 5 times with HEMG buffer and immunoblotted with anti-FLT3 antibody after SDS-PAGE. In the FLT3 dephosphorylation reaction, 5 μl of synthetic FLT3 was incubated with 5 U of calf intestine alkaline phosphatase (New England Biolabs, Beverly, Mass.) In a 10 μl reaction at 37 ° C for 1 hour prior to the binding experiment. It was done.

Suppression of Lyn expression by siRNA
Two predesigned siRNAs (siRNA IDs: 156196 and 156197) targeting Lyn exon 5 or exons 7 and 8 were purchased from Ambion (Austin, TX). BLOCK-iT Fluorescent Oligo ™ was purchased from Invitrogen and used as a control. A mixture of 2 Lyn siRNAs (1.5 μg each) or 3 μg control siRNA was transiently introduced into the cells by the Nucleofector system (amaxa biosystems, Gaithersburg, MD) as recommended by the manufacturer.

Administration of PP2 to living body 8 weeks old female C3H / HeNCrj mice were purchased from Charles River Japan Inc. (Atsugi, Japan). They were bred under standard conditions according to the guidelines of the Nagoya University Animal Experiment Facility. One million FLT3 / ITD-32D cells or Wt FLT3-32D cells were administered subcutaneously on the back of mice. PP2 administration was started from the time indicated in FIG. PP2 dosage was determined with reference to previous reports (Nam, JS, 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 × D) / 2
[D (mm) and D (mm) are the shortest and longest diameters of the tumor].

result
Tyrosine phosphorylation of Lyn in FLT3 / ITD-introduced 32D cells
It is known that FLT3 / ITD-32D exhibits IL-3 independent proliferation and STAT5 exhibits constitutive activation in the cell (Hayakawa, F., et al., (2000). Oncogene, 19,624-31). Therefore, paying attention to the pathway from FLT3 / ITD to STAT5, we investigated the phosphorylation of SFK. The results are shown in FIG. After 24 hours of cytokine starvation, each cell was stimulated with IL-3 (IL3) or FLT3 ligand (FL) for 10 minutes or nothing (-). The cell lysate was prepared as described in the experimental method. (A) JAK2 phosphorylation. Using 10 μl of the lysate, SDS-PAGE was followed by immunoblotting (IB) with anti-phosphorylated JAK2 antibody (top photo) or anti-JKA2 antibody (bottom photo). (B) Lyn phosphorylation. Immunoprecipitation (IP) was performed with rabbit (R) anti-Lyn antibody using 100 μl of cell lysate. IB with anti-phosphotyrosine (pTyr) antibody (top photo) and mouse (M) anti-Lyn antibody (bottom photo) was performed using 90% and 10% of the immunoprecipitates, respectively. (C) Src phosphorylation. Similar to (B), anti-Src antibody was used for IP, anti-phosphotyrosine antibody and anti-Src antibody were used for IB.

  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 which SFK is phosphorylated among SFKs that are known to phosphorylate STAT5 in addition to JAK2, first, 32D cells into which wild type FLT3 or FLT3 / ITD were introduced (Wt When FLT3-32D and FLT3 / ITD-32D) were stimulated with cytokines or not, the phosphorylation of various SFKs was examined. In FLT3 / ITD-32D, Lyn was constantly phosphorylated. On the other hand, Wyn FLT3-32D did not show Lyn phosphorylation even when stimulated with FLT3 ligand (FIG. 1B). The phosphorylation status of another SFK, Src, was also examined, but there was no difference between the two cells with or without cytokines (Fig. 1C). This suggests that Lyn is an upstream factor of STAT5 in FLT3 / ITD signal transduction, and that the signal transduction is involved in the cell proliferation effect brought about by FLT3 / ITD. 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, the phosphorylation of Lyn by wild-type FLT3 was reported, but this fact was not observed in our experiment.

The binding of Lyn and FLT3 is clearly strong in FLT3 / ITD and depends on the phosphorylation of FLT3.
Next, in order to investigate whether the binding of FLT3 and Lyn was specific to FLT3 / ITD, coprecipitation experiments were performed in which anti-FLT3 antibody was immunoprecipitated and immunoblotted with anti-Lyn antibody. The results are shown in FIG. After culturing for 6 hours in a cytokine-starved state, the cells were cytokine-stimulated as described in FIG. 1, and the cell lysate was prepared. (A) Binding of FLT3 and Lyn in the cell. 100 μl of 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) (upper photo) or anti-FLT3 antibody (lower photo). (B) Specular coprecipitation experiments using anti-Lyn antibody (R) for immunoprecipitation and anti-FLT3 antibody or anti-Lyn antibody (M) for immunoblotting were performed in the same manner as (A). (C) In vitro binding experiment of FLT3 versus Lyn. Wild-type and mutant FLT3 were synthesized in vitro and their expression was confirmed by immunoblotting using anti-FLT3 antibody (data not shown). An equal amount (5 μl) of FLT3 protein was precipitated by 1 μg of GST (G) or glutathione beads conjugated with Lyn (L) fused to GST. The precipitate was immunoblotted with anti-FLT3 antibody after SDS-PAGE (left photo). The same FLT3 protein used in the binding experiments was immunoprecipitated with anti-FLT3 antibody and immunoblotted with anti-phosphotyrosine antibody or anti-FLT3 antibody after SDS-PAGE (right photo). (D) In vitro binding experiment after dephosphorylation. FLT3 synthesized in vitro was incubated with a dephosphorylation solution with or without calf intestinal alkaline phosphatase (CIP) as shown in the figure prior to the binding experiment. In vitro binding experiments and confirmation of the phosphorylation state of FLT3 were performed as described in (C). (E) The position and amino acid sequence of the tandem duplication part of schema LLT3 / ITD of various Lyn and FLT3 constructs are shown. The black circles of 4FWt FLT3 and 8F FLT3 / mean the same amino acid sequences as Wt FLT3 and FLT3 / ITD, respectively. (F) Identification of binding regions in Lyn. GST fusion proteins from 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 and 10% was immunoblotted with anti-GST antibody. (G) Identification of the binding site of FLT3. In vitro binding experiments and confirmation of phosphorylation status were performed as described in (C).

  The binding (coprecipitation) between Lyn and FLT3 was observed with FLT3 / ITD-32D, but not with Wt FLT3-32D even after FLT3 ligand stimulation (Fig. 2A). This mutation-specific binding of FLT3 and Lyn was also confirmed by specular coprecipitation experiments using anti-Lyn antibody for immunoprecipitation and anti-FLT3 antibody for immunoblotting (FIG. 2B). Furthermore, in order to investigate whether the binding was direct or via other factors, a binding experiment was conducted in a test tube. It was suggested that the N-terminal half containing the SH3 and SH2 regions of Lyn and the GST fusion protein bind to the synthetic FLT3 in vitro, and that both bind directly (Fig. 2C left panel). Of note, wild-type FLT3 was also much weaker than FLT3 / ITD, but showed binding to Lyn, and the mutation specificity in the binding between FLT3 and Lyn was not absolute. In many cases, receptor tyrosine kinase (RTK) and SFK binding is dependent on receptor tyrosine phosphorylation (Boggon, T.J. & Eck, M.J. (2004). Oncogene, 23, 7918-27). Therefore, we investigated the phosphorylation state of the synthetic FLT3 used in the in vitro binding experiment, and examined whether this affects the binding of FLT3 and Lyn in this system. FLT3 synthesized in a system using rabbit reticulocyte lysate caused autotyrosine phosphorylation, and the phosphorylation of FLT3 / ITD was much stronger than wild-type FLT3 (Fig. 2C right panel). This suggests that the binding between FLT3 and Lyn depends on the tyrosine phosphorylation of FLT3, and that the difference in the degree of phosphorylation of FLT3 may cause the difference in binding.

  In order to investigate the phosphorylation dependence of this binding, in vitro binding experiments were performed using dephosphorylated FLT3. As expected, FLT3 dephosphorylation resulted in loss of binding to Lyn in both wild-type and mutant FLT3, suggesting its dependence on FLT3 phosphorylation. In most cases, the SH2 region is involved in the recognition of phosphorylated tyrosine. To further clarify the dependence of this binding on FLT3 tyrosine phosphorylation, whether or not the SH2 region of Lyn is involved in this binding is examined. Examined. Furthermore, the influence of tyrosine-phenylalanine substitution in the parasial region, which is considered to be the most probable as the Lyn binding region of FLT3, on the binding of both was investigated. In vitro binding experiments using various fusion components of Lyn and GST as shown in Fig. 2E and synthetic FLT3 / ITD revealed the involvement of the Lyn SH2 region in this binding (Fig. 2F). . The FLT3 / ITD used has a tandem duplication of the part corresponding to 583-602 amino acids of wild-type FLT3 in the parasite region. Substituting phenylalanine for 4 and 8 tyrosines in this region relative to wild type FLT3 and FLT3 / ITD (named 4F substitution and 8F substitution, respectively), the resulting mutant FLT3 was 4F Wt FLT3 and 8F FLT3, respectively. It was named / ITD (Figure 2E). In an in vitro binding experiment, 4F Wt FLT3 and 8F FLT3 / ITD showed attenuated binding to Lyn (approximately 30% of Wt FLT3 and FLT3 / ITD, respectively) (left panel in FIG. 2G). In addition, substitution of 4F and 8F significantly reduced tyrosine phosphorylation of wild-type FLT3 and FLT3 / ITD (Fig. 2G right panel). These data suggest that the substituted tyrosine is a phosphorylation site in the autophosphorylation of wild type FLT3 and FLT3 / ITD, and the phosphorylation of these tyrosine is recognized in the SH2 region of Lyn. The binding between wild-type and mutant FLT3 and Lyn shown in FIGS. 2A-G, the identification of the binding site, and the dependence of the binding on FLT3 phosphorylation were all clarified for the first time in the present invention.

Inhibition of specific Lyn signal by siRNA specifically suppressed cell growth by FLT3 / ITD.
We investigated the significance of Lyn activation in cytokine-independent growth by FLT3 / ITD by suppressing Lyn expression with siRNA designed based on Lyn-specific DNA sequences. The results are shown in FIG. Suppression of Lyn expression by siRNA. One million FLT3 / ITD-32D cells cultured in the presence of IL-3 were transfected with 3 μg of control siRNA (C) or 1.5 μg of two Lyn siRNAs using the Nucleofector system. Twenty-four hours after transfection, the cells were washed and placed in cytokine starvation for 24 hours. Then, the cell lysate was prepared, and after SDS-PAGE, immunoblotting with antibodies against Lyn, c-Src, Fyn, Lck, c-Yes and Hck was performed as shown in the figure. (B) Specific inhibition of STAT5. The same lysate as in (A) was used for immunoblotting with phosphorylation specific antibodies as shown. The expression of each signaling factor other than SFK was confirmed by immunoblotting with the corresponding non-phosphorylated antibody (data not shown). (C), (D) Effect of Lyn expression suppression by siRNA on the proliferation of various 32D cells introduced with FLT3. Wt FLT3-32D and FLT3 / ITD-32D were cultured in the presence of IL-3 and introduced siRNA as described in (A). Cells were washed 24 hours after transfection and reconstituted at a density of 2 × 10 ⁵ cells / ml with ⁵ ng / ml IL-3 (IL3), FLT3 ligand (FL) or no cytokine (-) culture as shown. Suspended (Time 0). The number of viable cells at each time point was measured by trypan blue exclusion method, and a graph was created by calculating the ratio to the value at Time 0 time point (2 × 10 ⁵ cells / ml). Each experiment was performed twice, and the mean and standard deviation are shown.

  In FLT3 / ITD-32D, it was confirmed by immunoblotting that Lyn expression was suppressed by about 90% by Lyn siRNA. Furthermore, the specificity of expression suppression was also confirmed by immunoblotting. That is, the expression of five SFKs other than Lyn was not clearly affected (FIG. 3A). The effect of Lyn expression suppression on FLT3 / ITD signaling was examined by immunoblotting with phosphorylation specific antibodies against each signaling factor. The phosphorylation of SFK markedly decreased to about 20%. Considering that the expression of SFKs other than Lyn was not affected, this decrease in SFK phosphorylation appears to be caused by suppression of Lyn expression. This indicates that phosphorylation detected by anti-phosphorylated SFK antibody is mainly Lyn phosphorylation in FLT3 / ITD-32D cells, and this antibody detects Lyn phosphorylation in FLT3 / ITD-32D cells It is possible to use it. STAT5 phosphorylation decreased to about 50% of control, but MAP kinase, Akt, and Shc phosphorylation was not affected (FIG. 3B). As shown here, phosphorylation recognized by anti-phosphorylated SFK antibody is only shown to be significantly (80% or more) reduced by Lyn-specific expression suppression (80% or more). It can be said that it is phosphorylation of Lyn. In Robinson et al.'S previous report, no such experiment was conducted, so it is unclear which phosphorylation the observed phosphorylation was.

Inhibition of specific STAT5 phosphorylation by suppressing Lyn expression suggests that Lyn is an upstream factor of STAT5 in the FLT3 / ITD signaling pathway.
The effect of Lyn siRNA on the proliferation of FLD3-introduced 32D was examined. In the absence of IL-3, FLT3 / ITD-32D proliferation was markedly suppressed by Lyn siRNA (Fig. 3D). On the other hand, in the presence of IL-3, 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 (FIGS. 3C and 3D). These results suggest that Lyn is more important in cell proliferation induced by FLT3 / ITD signal than cell proliferation induced by wild-type FLT3 signal or IL-3 signal. In addition, since SFK inhibitors such as PP1 and PP2 used in the above-mentioned report of Robinson et al. Are not clear in SFK, even if the growth of cells is suppressed by SFK inhibitor, It is not possible to identify which SFK is responsible for it, which SFK is important for cell proliferation. As shown in the present invention, it is possible to identify that Lyn is important for the first time by suppressing Lyn-specific expression. That is, this is the first time that Lyn has been identified to be important for cell growth in cells expressing FLT3 mutants.

Inhibition of FLT3 / ITD-32D 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 was examined. Both cell lines were cultured for 24 hours with various concentrations of PP2. The results are shown in FIG. (A) Concentration-dependent growth inhibition by PP2. Wt FLT3-32D (Wt) or FLT3 / ITD-32D (Mut) was cultured for 24 hours with the addition of PP2 at the indicated concentrations in the presence / absence of IL-3. 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 μM in%. Each experiment was performed in triplicate and the mean and standard deviation are shown. (B) Growth curve of cells treated with PP2. FLT3 / ITD-32D was cultured by adding IL-3 (2 ng / ml) and PP2 (10 μM) in a combination as shown. Viable cell count and graph creation are the same as in Fig. 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 μM PP2 was added to FLT3 / ITD-32D cultured in the presence / absence of IL-3. At the time indicated, the cell lysate was prepared as described above. FLT3 phosphorylation was examined as in FIG. 1B except that anti-FLT3 antibody was used for immunoprecipitation. The phosphorylation of other signaling factors was examined as in FIG. 3B. It was confirmed by immunoblotting with a corresponding non-phosphorylated antibody that the expression of each factor or the efficiency of FLT3 immunoprecipitation was equivalent between each time point.

The growth inhibitory effect was clearly concentration-dependent and was strong in FLT3 / ITD-32D cultured in the absence of IL-3. The GI ₅₀ for FLT3 / ITD-32D in the absence of IL-3 was approximately one third that of Wt FLT3-32D and FLT3 / ITD-32D in the presence of IL-3 (5 μM vs. 17.7 μM (FIG. 4A). The cell growth curves of FLT3 / ITD-32D with addition of 10 μM PP2 were also compared in the presence and absence of IL-3. In the absence of IL-3, the growth of FLT3 / ITD-32D was completely inhibited by PP2. However, some suppression of proliferation was observed even in the presence of IL-3 (FIG. 4B). Cell growth suppression by PP2 is considered to be more effective against FLT3 / ITD than that induced by IL-3, which indicates that PP2 is resistant to cancer caused by FLT3 / ITD. It has the potential to act as a tumor drug. Next, the effect of PP2 on phosphorylation of signaling factors 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 μM PP2 (FIG. 4C, upper panel). However, in the absence of IL-3, phosphorylation of SFK, which mainly consists of phosphorylation of Lyn, disappeared rapidly within 1 hour, and phosphorylation of STAT5 also disappeared within 6 hours. Mild attenuation of MAP kinase phosphorylation was observed 12 hours later, and phosphorylation of Akt and Shc was not affected (Fig. 4C). JAK2 phosphorylation was not observed at any time (data not shown). On the other hand, JAK2 phosphorylation was observed in the presence of IL-3, which was slightly suppressed after 12 hours. In line with the remaining phosphorylation of JAK2, the phosphorylation of STAT5 also remained. Attenuation of MAP kinase phosphorylation was observed to the same extent as in the absence of IL-3 (FIG. 4D). Akt and Shc phosphorylation were not affected (data not shown). From these results, it was not possible to determine whether the phosphorylation inhibition of the signal transduction factor was a direct effect of Lyn inhibition, SFK inhibition other than Lyn, or inhibition of kinases other than SFK. However, signal inhibition by PP2 appears to occur more strongly with STAT5, which supports the hypothesis that Lyn is an upstream factor of STAT5.

Therapeutic effect of PP2 on tumor with FLT3 / ITD in mouse model
In order to examine the possibility of the SFK inhibitor as a therapeutic drug for cancer caused by FLT3 / ITD, a mouse model of a tumor developed by FLT3 / ITD developed earlier (a mouse not irradiated with FLT3 / ITD-32D) (Zhao et al., (2000). Leukemia, 14, 374-8.). The results are shown in FIG. Six mice were inoculated subcutaneously with FLT3 / ITD-32D on day 1 and intraperitoneally from day 4 to day 63 only 5 μg or 10 μg PP2 or DMSO dissolved in DMSO twice a week Was administered. 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 μg or 10 μg PP2 or DMSO alone dissolved in 100 μl DMSO twice a week, intraperitoneally (ip.) From day 4 to day 63. Control mice received 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 the 25th day to the 63rd day, the indicated amount of PP2 dissolved in 100 μl of DMSO was intraperitoneally administered twice a week.

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

  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 administered PP2 or DMSO after tumors developed (day 25). In control mice, subcutaneous tumors grew rapidly and all mice died by day 45. On the other hand, in the group to which 30 μg of PP2 was administered, tumor progression was slow, and all mice were still alive at day 63. Furthermore, in the group administered with 60 μg PP2, stronger growth inhibition was observed. That is, the tumor was reduced by the 35th day until it could not be detected (FIG. 5B). Both mice in this group continued to survive 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.

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

Claims (8)

A method for identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, wherein the active mutant FLT3 is contacted with Lyn in the presence of the test substance, and the test substance inhibits binding between the active mutant FLT3 and Lyn. And / or determining whether to inhibit phosphorylation of Lyn in cells expressing the active mutant FLT3. A method of identifying a candidate substance for a therapeutic agent for acute myeloid leukemia, comprising contacting a test substance with a cell expressing Lyn, whether the test substance inhibits Lyn expression, and / or STAT5 by Lyn Determining whether to inhibit phosphorylation. A substance selected from the group consisting of an antisense oligonucleotide for Lyn gene, a ribozyme for Lyn gene, siRNA for Lyn gene, PP1, PP2, SU6656, staurosporine, NS-187, KRX-123, and BMS-354825 A therapeutic agent for acute myeloid leukemia containing as an active ingredient. A substance that inhibits the binding of active mutant FLT3 to Lyn, a substance that inhibits phosphorylation of Lyn in cells that express active mutant FLT3, a substance that inhibits the expression of Lyn, and a phosphorylation of STAT5 by Lyn A therapeutic agent for acute myeloid leukemia comprising a substance selected from the group consisting of substances to be treated as an active ingredient. A method of treating acute myeloid leukemia, comprising inhibiting binding of active mutant FLT3 to Lyn and / or inhibiting phosphorylation of Lyn in cells expressing active mutant FLT3 . A method of treating acute myeloid leukemia comprising inhibiting Lyn expression and / or inhibiting STAT5 phosphorylation by Lyn. A method for treating acute myeloid leukemia, in which an antisense oligonucleotide for Lyn gene, a ribozyme for Lyn gene, siRNA for Lyn gene, PP1, PP2, SU6656, staurosporine, NS Administering a substance selected from the group consisting of -187, KRX-123, and BMS-354825. A method for treating acute myeloid leukemia, which inhibits the phosphorylation of Lyn in cells expressing active mutant FLT3, a substance that inhibits the binding of active mutant FLT3 and Lyn to patients in need of treatment Administering a substance selected from the group consisting of a substance that inhibits the expression of Lyn, and a substance that inhibits phosphorylation of STAT5 by Lyn.