WO2007142139A1 - Procédé d'identification de médicaments candidats pour le traitement de la leucémie myéloïde aiguë - Google Patents

Procédé d'identification de médicaments candidats pour le traitement de la leucémie myéloïde aiguë Download PDF

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WO2007142139A1
WO2007142139A1 PCT/JP2007/061190 JP2007061190W WO2007142139A1 WO 2007142139 A1 WO2007142139 A1 WO 2007142139A1 JP 2007061190 W JP2007061190 W JP 2007061190W WO 2007142139 A1 WO2007142139 A1 WO 2007142139A1
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lyn
flt3
myeloid leukemia
acute myeloid
phosphorylation
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PCT/JP2007/061190
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English (en)
Japanese (ja)
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Tomoki Naoe
Fumihiko Hayakawa
Mitsunori Okamoto
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National University Corporation Nagoya University
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Priority to JP2008520538A priority Critical patent/JP5246776B2/ja
Publication of WO2007142139A1 publication Critical patent/WO2007142139A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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 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.
  • 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.
  • the active mutation FLT3 was frequently observed in adult acute myeloid leukemia (AML) cases.
  • AML acute myeloid leukemia
  • the receptor and its downstream signaling factor are constitutively activated and cell proliferation is promoted.
  • 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) ⁇ 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.
  • 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) ⁇ eukemia, ll, 1442-6; Kiyoi, H., et al., (1999) .Blood, 93, 3074-80;
  • 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
  • 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! / ,!
  • 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.
  • SFK Src family kinases
  • Non-patent document 1 Yokota, S., et al., (1997) ⁇ 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) ⁇ 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 Thomasger, S., et al., (2002) .Blood, 100, 59-66
  • Non-Patent Document 10 Shih, L.Y., et al., (2004) ⁇ 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
  • 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 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. .
  • 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.
  • 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.
  • 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;
  • 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-
  • 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.
  • 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,
  • 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.
  • 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.
  • 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:
  • 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.
  • 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:
  • FIG. 1 shows phosphorylation of SFK in FLT3 / ITD-introduced 32D cells.
  • Figure 2 shows the coupling of FLT3 and Lyn.
  • Fig. 3 shows phosphorylation of STAT5 and inhibition of cell proliferation by FLT3 / ITD when Lyn expression was suppressed by siRNA.
  • Figure 4 shows the effect of SFK inhibitors on FLT3 / ITD signaling and cell proliferation.
  • FIG. 5 shows inhibition of proliferation of FLT3 / ITD-32D cells in C3H / HeNCj mice by PP2.
  • 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.
  • FLT3 / ITD and Lyn are specifically bound to ITD mutation in the cell, and Lyn phosphonate is associated with it.
  • Lyn phosphonate is associated with it.
  • the binding is more affinity to FLT3 / ITD than wild-type FLT3 and is dependent on FLT3 phosphate.
  • 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.
  • 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.
  • 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.
  • 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.
  • extracts from natural products such as bacteria, fungi, algae, plants, animals, and partially purified products may be used as test substances.
  • 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.
  • RNAi RNA interference
  • 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 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a carrier used in the preparation any of those commonly used in the pharmaceutical field can be used.
  • sterile water, physiological saline, excipient, stabilizer, antioxidant, buffer, surfactant Agents and binders are preferably used.
  • 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.
  • 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.
  • 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.
  • 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.
  • FBS urinary fetal serum
  • FLT3 ligand R & D Systems, Minneapolis, Minn.
  • 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)
  • FLT3 / pcDNA and 8F FLT3 / ITD / pcDNA were prepared by PCR mutagenesis using the QuikChange TM site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).
  • 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).
  • lysis buffer 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, l% Nonidet P-40, 0.1% dodecyl sulfate.
  • Immunoprecipitation and immunoblotting were performed as previously reported (Hong, SH & Privalsky, M ⁇ . (2000). Mol Cell
  • siRNAs targeting Lyn exon 5 or exons 7 and 8 Two predesigned siRNAs (siRNAs targeting Lyn exon 5 or exons 7 and 8)
  • 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 ⁇ g each) or 3 ⁇ g 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.
  • mice 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:
  • FLT3 / ITD-32D shows IL-3 independent proliferation
  • 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.
  • FLT3 protein Amount (5 ⁇ l) of FLT3 protein was precipitated with 1 ⁇ g 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.
  • 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.
  • 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). .
  • siRNA specifically inhibits cell multiplication by FLT3 / ITD
  • Lyn siRNA 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.
  • 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.
  • the lower Wt FLT3-32D and FLT3 / ITD-32D were about one third (5 ⁇ vs. 17.7 ⁇ .) (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 ⁇ were also compared.
  • FLT3 / ITD-32D proliferation was completely inhibited by ⁇ 2.
  • some suppression of proliferation was observed even in the presence of IL-3 (Fig. 4).
  • 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).
  • 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.
  • 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.
  • the present invention is useful for screening candidate substances for therapeutic agents for acute myeloid leukemia and for treating acute myeloid leukemia.

Abstract

L'invention concerne un procédé d'identification d'un médicament candidat pour le traitement de la leucémie myéloïde aiguë, comprenant les étapes consistant à mettre un mutant de FLT3 en contact avec Lyn en présence d'une substance de test et à rechercher si la substance de test inhibe ou non la liaison du mutant de FLT3 activé à Lyn et/ou si la substance de test inhibe ou non la phosphorylation de Lyn dans une cellule exprimant le mutant de FLT3 activé. L'invention concerne également un procédé pour le traitement de la leucémie myéloïde aiguë, comprenant les étapes consistant à mettre une substance de test en contact avec une cellule exprimant Lyn et à rechercher si la substance de test inhibe ou non l'expression de Lyn et/ou si la substance de test inhibe ou non la phosphorylation de STAT5 par Lyn. Une substance identifiée par le procédé ci-dessus est utile comme principe actif d'un médicament destiné à traiter la leucémie myéloïde aiguë.
PCT/JP2007/061190 2006-06-02 2007-06-01 Procédé d'identification de médicaments candidats pour le traitement de la leucémie myéloïde aiguë WO2007142139A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2047849A1 (fr) * 2007-10-08 2009-04-15 KTB Tumorforschungsgesellschaft mbH Utilisation d'imides d'indolocarbazole en tant qu'inhibiteurs de protéine kinase pour le traitement des tumeurs solides et hématologiques
WO2009047216A2 (fr) * 2007-10-08 2009-04-16 Ktb Tumorforschungsgesellschaft Mbh Utilisation d'indolocarbazole-imides en tant qu'inhibiteurs sélectifs de protéine kinases
WO2009047216A3 (fr) * 2007-10-08 2009-10-01 Ktb Tumorforschungsgesellschaft Mbh Utilisation d'indolocarbazole-imides en tant qu'inhibiteurs sélectifs de protéine kinases
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CN116602947A (zh) * 2023-05-18 2023-08-18 苏州大学 一种小分子化合物及其在制备治疗flt3突变型白血病药物中的应用
CN116602947B (zh) * 2023-05-18 2023-12-08 苏州大学 一种小分子化合物及其在制备治疗flt3突变型白血病药物中的应用

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