JP2005281183A - Therapeutic agent for acute leukemia by using pml activating agent, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a differentiation inducing drug for a leukemic cell, and a therapeutic agent for leukemia containing the differentiation inducing drug. <P>SOLUTION: It is discovered that the function of an AML (acute myelocytic leukemia) 1 complex is activated by dephosphorylation of a PML (promyelocytic leukemia) protein which is a constituent factor of the AML1 complex, and a differential protein dephosphorylation enzyme PMLK (kinase) for controlling the phosphorylation of the PML protein is identified. The inhibitor of the PMLK is considered to be able to induce the differentiation of the leukemic cell by activating the AML1 complex through the dephosphorylation of the PML protein. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a PML protein expression activator or function activator, or a leukemia cell differentiation inducer comprising a PMLK protein expression suppressor or function suppressor. The present invention also relates to a screening method for candidate compounds for leukemia therapeutic agents.

  In human leukemia and lymphoma, specific chromosomal translocations are frequently observed, and the resulting expression of fusion gene products and abnormal expression of oncogenes are involved in the development of human leukemia and lymphoma (Non-patent Document 1). reference). AML1 gene is the most frequently targeted chromosomal translocation and identified as a gene located at the translocation point of chromosome 21 in the t (8; 21) translocation frequently found in the M2 subtype of acute myeloid leukemia (See Non-Patent Document 2). In addition, the AML1 gene has so far been t (3; 21) (see non-patent documents 3 and 4), t (12; 21) (see non-patent documents 5 and 6), t (16; 21) (non-patent document). 7 and 8) have been shown to be involved in various chromosomal translocations. The AML1 gene is also mutated in acute myeloid leukemia (AML) without chromosomal translocation, myelodysplastic syndrome (MDS), and familial thrombocytopenia (FPD) that develops leukemia frequently. (See Non-Patent Documents 9 to 11).

  AML1 protein regulates the expression of many hematopoietic cell-specific genes by forming heterodimers with CBFβ protein and binding to specific DNA sequences. It has been clarified from analysis using gene-disrupted mice that both AML1 and CBFβ are essential for adult hematopoiesis (see Non-Patent Documents 12 to 16). AML1 functions by binding to other transcription factors, or transcription coupling factors such as p300 / CBP (see Non-Patent Document 17) or MOZ (see Non-Patent Document 18), and functions as a factor that integrates transcription complexes. Has been proposed. It should be noted that many of the factors that form complexes with AML1 are targets for chromosomal translocations found in leukemia. CBFβ is involved in inv (16) (see non-patent document 19), and p300 is a t (8; 22) translocation (see non-patent documents 20 and 21) or t (11; 22) translocation (non-patent document 21). CBP is t (8; 16) translocation (see non-patent document 23) or t (11; 16) translocation (see non-patent documents 24-26), and MOZ is t (8; 16) translocation. (See Non-Patent Document 23), inv (8) (see Non-Patent Document 27) or t (8; 22) translocation (see Non-Patent Documents 20 and 21). In total, including AML1 mutations, 40-50% of acute myeloid leukemias showed abnormalities in one of the components of the AML1 complex, so the AML1 complex is a major chromosomal translocation in acute leukemia. Considered a target.

  The promyelocytic leukemia (PML) gene is involved in the t (15; 17) translocation found in most acute promyelocytic leukemia (see Non-Patent Documents 28 to 33). Analysis of PML gene-deficient mice suggests that PML controls the differentiation, cell proliferation, and canceration of hematopoietic progenitor cells (see Non-Patent Document 34), but little is known about the mechanism of action. PML is involved in cancer suppressive functions such as apoptosis induction, growth arrest, and cellular aging (see Non-Patent Document 35). PML, along with many other proteins, is localized in a spotted region in the nucleus. Such proteins co-localized with PML include Sp100, p53, pRb, Daxx, CBP, etc., suggesting that PML regulates various nuclear processes such as gene expression and genome stability. ing. Although it is known that many isoforms are expressed by splicing different from the PML gene (see Non-Patent Documents 36 and 37), there are many unclear points about specific functions of each isoform.

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Kitabayashi I, 4 other authors, "Activation of AML1-mediated transcription by MOZ and inhibition by the MOZ-CBP fusion protein.", EMBO J., 2001 Dec 17, Vol. 20 (24), p.7184-96 Liu P, 7 others, "Fusion between transcription factor CBF beta / PEBP2 beta and a myosin heavy chain in acute myeloid leukemia.", Science, 1993 Aug 20, Vol.261 (5124), p.1041-4 Chaffanet M, G.L., 4 other authors, "MOZ is fused to p300 in an acute monocytic leukemia with t (8; 22).", Genes Chromosomes Cancer, 2000, Vol. 28, p.138-144 Kitabayashi I, 7 others, "Fusion of MOZ and p300 histone acetyltransferases in acute monocytic leukemia with at (8; 22) (p11; q13) chromosome translocation.", Leukemia, 2001 Jan, Vol. 15 (1), p. .89-94 Ida, K., `` Adenoviral E1A-associated protein p300 is involved in acute myeloid leukemia with t (11; 22) (q23; q13). '', Blood, 1997, Vol. 90, p.4699-704 Borrow, J., “The translocation t (8; 16) (p11; p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the CREB-binding protein.”, Nat Genet, 1996, Vol. 14, p. 33 -41 Taki T, 3 other authors, "The t (11; 16) (q23; p13) translocation in myelodysplastic syndrome fuses the MLL gene to the CBP gene.", Blood, 1997, Vol. 89, p.3945-3950 Sobulo, OM, "MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with at (11; 16) (q23; p13.3).", Proc Natl Acad Sci USA, 1997, Vol. .94, p.8732-7 Satake N, IY, 6 other authors, "Novel MLL-CBP fusion transcript in therapy-related chronic myelomonocytic leukemia with at (11; 16) (q23; p13) chromosome translocation.", Genes Chromosomes Cancer, 1997, Vol.20 , P.60-63 Carapeti M, 3 other authors, “A novel fusion between MOZ and the nuclear receptor coactivator TIF2 in acute myeloid leukemia.”, Blood, 1998 May 1, Vol. 91 (9), p.3127-33 de The H, 4 authors, “The t (15; 17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus.” Nature, 1990, Vol.347, p.558-561 de The H, 5 other authors, “The PML-RAR alpha fusion mRNA generated by the t (15; 17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.”, Cell, 1991, Vol. 66, p.675 -684 Goddard AD, 3 other authors, "Characterization of a zinc finger gene disrupted by the t (15; 17) in acute promyelocytic leukemia.", Science, 1991, Vol. 254, p.1371-1374 Kakizuka A, 7 others, "Chromosomal translocation t (15; 17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML.", Cell, 1991, Vol. 66, p.663-674 Pandolfi PP, 6 authors, “Structure and origin of the acute promyelocytic leukemia myl / RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties”, Oncogene, 1991, Vol. 6, p.1285-1292 Kastner P, 8 other authors, “Structure, localization and transcriptional properties of two classes of retinoic acid receptor alpha fusion proteins in acute promyelocytic leukemia (APL): structural similarities with a new family of oncoproteins.”, EMBO J., 1992, Vol.11, p.629-642 Wang ZG, 7 authors, "Role of PML in cell growth and the retinoic acid pathway.", Science, 1998, Vol. 279, p.1547-1551 Salomoni P and Pandolfi PP., `` The role of PML in tumor suppression. 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  An object of the present invention is to elucidate the mechanism of the differentiation induction phenomenon of leukemia cells by PML protein, and based on the obtained knowledge, a novel leukemia cell differentiation inducer and the differentiation inducer The issue is to provide therapeutic drugs for leukemia.

  The present inventor has intensively studied to solve the above problems. As a therapeutic agent for leukemia, a molecular target reagent that suppresses the activity of a fusion protein expressed specifically in leukemia is considered first. Gleevec (Novatis Pharma Co., Ltd.), which has already been clinically applied, specifically inhibits BCR-ABL fusion tyrosine kinase expressed in 90% or more of chronic myeloid leukemia. However, AML1-MTG8, CBFβ-MYH11, and PML-RARα expression, which is considered to be the most common in acute myeloid leukemia, is about 10-20% of the total in acute leukemia. These fusion proteins were difficult to become common molecular targets.

  Therefore, the present inventors focused on a reagent that activates the AML1 complex, which is a major target of chromosome translocation, as a therapeutic agent that can be expected next. AML1-MTG8, CBFβ-MYH11, and MOZ-CBP whose functions have been analyzed so far have been shown to act dominantly on AML1 protein and suppress transcriptional activation by AML1 protein. In many leukemias, alleles are normal, so normal AML1 complex may also coexist, and a reagent that reactivates AML1 complex repressed by the fusion protein is applied to the treatment of acute leukemia Can be expected. In fact, the present inventor has shown that differentiation of leukemia cells is promoted by forced expression of the AML1 gene.

  In addition, the present inventor has found that AML1 protein constitutes a complex with PML protein (AML1-PML complex), and the function of the complex is activated by dephosphorylation of PML protein as a constituent factor. I found out that Furthermore, the present inventor has succeeded in identifying a specific protein kinase, PMLK, that controls phosphorylation of PML protein.

  An inhibitor of PMLK is thought to be able to induce differentiation of leukemia cells by activating the AML1-PML complex through dephosphorylation of PML protein. That is, a PML protein functional activator or a PMLK protein functional inhibitor is expected to be a leukemia cell differentiation inducer. The knowledge about these PML protein and PMLK protein was discovered for the first time by the present inventors.

Although it has been reported that JNK protein (PMLK protein) is involved in the differentiation of mammalian stem cells (patent international publication WO03102151), the phosphorylation substrate of JNK has not been clarified so far. The mechanism of cell differentiation induction was still unclear. That is, the knowledge about phosphorylation using the above-mentioned PML as a substrate found by the present inventors has not been known so far. Based on the findings discovered by the present inventors, a leukemia cell differentiation inducer or leukemia therapeutic agent targeting phosphorylation of PML protein has been provided for the first time. Furthermore, by using the phosphorylation state of PML protein as an index, it has become possible to easily screen for a leukemia cell differentiation inducer or a leukemia therapeutic agent.
As described above, the present inventors have succeeded in elucidating the mechanism of differentiation induction of leukemia cells by PMLK protein, and have completed the invention of a differentiation inducer for leukemia cells.

The present invention relates to a PML protein expression activator or function activator, or a leukemia cell differentiation inducer comprising a PMLK protein expression inhibitor or function inhibitor, and a leukemia therapeutic agent comprising the differentiation inducer. More specifically,
[1] An agent for inducing differentiation of leukemia cells, comprising a PML protein expression activator or function activator,
[2] The agent for inducing differentiation of leukemia cells according to [1], wherein the functional activator of PML protein is an activator of PML protein phosphatase or an inhibitor of PML protein phosphatase,
[3] A site on the PML protein that suppresses phosphorylation of the serine sites at positions 505, 518, 527, and / or 530 of the amino acid sequence shown in SEQ ID NO: 2, or the serine site An agent for inducing differentiation of leukemia cells, comprising a substance that promotes dephosphorylation of
[4] An agent for inducing differentiation of leukemia cells, comprising a PMLK protein expression inhibitor or function inhibitor,
[5] The differentiation inducer for leukemia cells according to [4], wherein the PMLK protein expression inhibitor is a compound selected from the group consisting of the following (a) to (c):
(A) An antisense nucleic acid for the transcription product of the PMLK gene or a part thereof (b) A nucleic acid having ribozyme activity that specifically cleaves the transcription product of the PMLK gene (c) It has an inhibitory action by the RNAi effect on the expression of the PMLK gene A therapeutic agent for leukemia, comprising as an active ingredient the differentiation-inducing agent for leukemia cells according to any of nucleic acids [6] [1] to [5],
[7] A screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c):
(A) a step of contacting a test compound with a cell expressing the PMLK gene (b) a step of measuring the expression level or activity of the PMLK protein in the cell (c) as compared with a case where the test compound is not contacted, A step of selecting a compound that reduces the expression level or activity [8] a screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c):
(A) the step of contacting the PML protein and the test compound (b) the step of measuring the phosphorylation state of the PML protein (c) the compound that reduces the degree of phosphorylation compared to the case where the test compound is not contacted [9] a screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c):
(A) Step of contacting PML protein, PMLK protein and test compound (b) Step of measuring phosphorylation activity of PMLK protein using PML protein as substrate (c) Compared with the case of not contacting test compound And [10] selecting a compound that decreases the phosphorylation activity. The phosphorylation site in the PML protein is the amino acid sequence of the PML protein represented by SEQ ID NO: 2 at positions 505, 518, 527, and / or Or the screening method according to [8] or [9], which is a serine site at position 530,
Is to provide.

  In many leukemias, the allele is normal, so normal AML1-PML complexes may also coexist, and reagents that reactivate AML1-PML complexes suppressed by fusion proteins are Application to treatment can be expected. In fact, it has been shown that forced expression of the AML1 gene promotes differentiation of leukemia cells. In addition, PMLK inhibitors have been shown to induce differentiation of bone marrow cells into neutrophils. From these results, it is considered that a compound that activates the expression or function of PML or a compound that inhibits the expression or function of PMLK is actually effective in treating leukemia.

  Further, based on the findings found by the present inventor, a novel screening method for a therapeutic agent for leukemia has been provided. By the screening method of the present invention, it is possible to efficiently obtain a compound having a leukemia therapeutic effect by a novel mechanism of action.

For the first time, the inventors have found that the function of the AML1-PML complex is activated by dephosphorylation of PML protein. In addition, PMLK, a PML protein-specific phosphorylase that controls phosphorylation of PML protein, was identified. That is, it is considered that a substance (compound) that suppresses phosphorylation of PML protein or a substance (compound) that promotes dephosphorylation of the protein activates AML1 protein and has an action of inducing differentiation of leukemia cells.
As described above, since PMLK protein has phosphorylase activity using PML protein as a substrate, compounds that inhibit PMLK expression or function activate AML1-PML complex through dephosphorylation of PML protein. It is considered effective for the treatment of leukemia by inducing differentiation of leukemia cells.

  In general, leukemia is a disease in which cells that are the basis of blood cells called hematopoietic stem cells in the bone marrow or progenitor cells derived therefrom are abnormally proliferated. This is caused by mutations or chromosomal translocations in one or several genes, resulting in abnormal regulation of cell differentiation / proliferation / apoptosis (cell death), resulting in white blood cell proliferation in the bone marrow, Immature white blood cells called blasts increase in the blood. Under normal conditions, leukocytes have a function of protecting the body by attacking bacteria and viruses that have entered the body. Therefore, when leukemia occurs, the function of protecting the body is lost. In acute leukemia, only immature white blood cells are produced, and normal white blood cells, platelets and red blood cells are not produced at all, and the disease progresses rapidly.

  In the present invention, the term “leukemia cell” refers to bone marrow cells and peripheral blood cells derived from leukemia patients, cells that have been immortalized or immortalized and cultured in vitro, and introduction of genes into leukemia cells or cultured cells or leukemia It refers to cells that mimic leukemia induced by viral infection. Specific examples include cultured cells derived from leukemia such as K562, ML1, U937.

  The present inventor confirmed that the PML protein that forms a complex with the AML1 protein is activated, that is, the PML protein is dephosphorylated, and the function of the PML protein is activated. As a result, the AML1 protein is activated. It was found that differentiation induction of leukemia cells was caused. That is, the PML protein itself, or a PML protein expression activator or functional activator is considered to be an AML1 protein activator. The AML1 protein activator is considered to have an action of inducing differentiation of leukemia cells, and is considered to be a therapeutic agent for leukemia, for example.

The present invention first relates to an AML1 protein activator containing at least one of any of the following (a) to (c). (In the present invention, the following proteins (a) and (b) may be collectively referred to as “PML protein”).
(A) PML protein (b) A PML protein variant comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the PML protein, and is functionally equivalent to the PML protein (C) DNA encoding the protein according to (a) or (b), or a vector expressing the protein

  The PML protein in the present invention is usually (endogenous) protein derived from a biological species having leukemia cells to be induced for differentiation. The PML protein of the present invention is preferably a human PML protein, but the species from which it is derived is not particularly limited, and examples thereof include PML proteins of mammals such as mice. As an example, the amino acid sequence of human PML protein is shown in SEQ ID NO: 2, and the DNA sequence encoding the protein is shown in SEQ ID NO: 1.

The “PML protein” in the present invention can be prepared not only as a natural protein but also as a recombinant protein using gene recombination technology. A natural protein can be prepared by, for example, a method using affinity chromatography using an antibody against PML protein against an extract of cells (tissue) considered to express PML protein. On the other hand, a recombinant protein can be prepared by culturing cells transformed with a DNA encoding a PML protein.
In the present invention, the “functionally equivalent protein” is a protein having the same activity as that revealed in the PML protein. Examples of the “activity” include an activity of AML protein binding (interacting activity). Measurement of the binding activity (interaction activity) between proteins can be appropriately performed by those skilled in the art by a known method.

The PML protein in the present invention is, for example, 70% or more, desirably 90% or more, more desirably 95% or more homology to the amino acid sequence of the PML protein (for example, the sequence described in SEQ ID NO: 2). The protein having sex can be shown as a protein functionally equivalent to the PML protein. The amino acid modification in the present invention is usually within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and further preferably within 3 amino acids.
Examples of the functionally equivalent protein include a human PML protein variant, a homologue of a human PML protein in a non-human organism, and the like.

  Further, the DNA encoding the PML protein in the present invention may be chromosomal DNA or cDNA. Chromosomal DNA encoding PML protein can be obtained, for example, by preparing a library of chromosomal DNA from cells or the like and screening the library using a probe that hybridizes to DNA encoding PML protein. The cDNA encoding the PML protein can be extracted from an RNA sample extracted from a cell (tissue) that is thought to express the PML protein, and the RT-PCR method using primers that hybridize to the DNA encoding the PML protein. It can be obtained by gene amplification technology.

  The PML protein or DNA encoding the protein in the present invention may be a mutant having a modified base sequence or amino sequence as long as it is functionally equivalent to the PML protein. Such mutants may be natural or artificial. Methods for artificially preparing mutants are known to those skilled in the art. For example, Kunkel method (Kunkel, TA et al., Methods Enzymol. 154, 367-382 (1987)), double primer method (Zoller, MJ and Smith, M., Methods Enzymol. 154, 329-350 (1987)) , Cassette mutation method (Wells, et al., Gene 34, 315-23 (1985)), megaprimer method (Sarkar, G. and Sommer, SS, Biotechniques 8, 404-407 (1990)) Yes.

In the present invention, as the AML1 activator, DNA encoding the protein described in the following (a) or (b) or a vector expressing the protein can be used.
(A) PML protein,
(B) A PML protein variant containing an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the PML protein, and is functionally equivalent to the PML protein

  The AML1 activator of the present invention is expected to be an agent for inducing differentiation of leukemia cells whose action mechanism is activation of AML1 or a therapeutic agent for leukemia.

  The DNA encoding the protein is preferably functionally linked to a promoter for efficient expression. As these promoters, the original PML gene promoter can be used, but besides this, various known promoters such as CMV promoter can be used. In addition, those skilled in the art can easily prepare vectors that express the above proteins using various known expression vectors.

  The above vector can also be used for gene therapy. Gene therapy refers to administration or treatment or prevention of a vector containing a DNA encoding a functional protein to a patient. Examples of vectors that can be used for gene therapy include, but are not limited to, adenovirus vectors (eg, pAdex1cw) and retrovirus vectors (eg, pZIPneo). General genetic manipulations such as insertion of DNA encoding the above protein into a vector can be performed according to conventional methods. In vivo administration may be carried out by ex vivo method, but in vivo method is preferred.

  The present invention also provides an AML1 protein activator and a leukemia cell differentiation inducer comprising a PML protein expression activator or function activator.

The “protein expression activator” in the present invention is a substance (compound) that significantly activates (increases) protein expression. The above “expression activation” of the present invention includes transcription activation of a gene encoding the protein and / or translation activation from a transcription product of the gene.
Examples of the PML protein expression activator of the present invention include substances (transcription activators, etc.) that bind to the transcriptional regulatory region (eg, promoter region) of the PML gene and promote the transcription of the gene. Can do.
In the present invention, the expression activity of the PML protein can be easily measured by those skilled in the art by methods well known to the art, such as Northern blotting and Western blotting.

  The PML protein function-activating substance is a substance that significantly activates the function of the PML protein. The inventors have found that PML protein is activated by dephosphorylation of PML protein. Therefore, examples of the functional activator of the PML protein of the present invention include a substance that suppresses phosphorylation of PML protein, an inhibitor of PML protein phosphorylase, or an activator of PML protein dephosphoryase Can do. An example of the phosphorylating enzyme is PMLK protein. That is, leukemia cell differentiation can also be induced by inhibiting the expression or function of PMLK protein. The dephosphorylating enzyme refers to an enzyme responsible for a reaction for removing a phosphate group on a PML protein.

  The PMLK protein is a phosphorylase using PML protein as a substrate, and usually phosphorylates serine sites at positions 505, 518, 527, and / or 530 on the PML protein. The present inventor has found that AML1 is activated by dephosphorylation of the site, and as a result, differentiation of leukemia cells is induced.

  Therefore, the dephosphorylating enzyme in the present invention is preferably dephosphorylated at the serine sites at positions 505, 518, 527, and / or 530 on the PML protein (amino acid sequence described in SEQ ID NO: 2). Those having an oxidizing activity are preferred. In addition, in the PML protein (homolog etc.) which consists of sequences other than sequence number: 2, the site | part phosphorylated may not necessarily correspond with the site | part shown by said numerical value. Therefore, in the present invention, the “site on the PML protein that can be phosphorylated” includes a serine site corresponding to the above site. Whether or not it is a “corresponding serine site” can be determined by those skilled in the art by performing homology alignment with the amino acid sequence shown in SEQ ID NO: 2, for example, the serine site at position 505 in SEQ ID NO: 2 In the PML protein homologue, etc., it is possible to know appropriately what site the serine corresponds to.

The present invention suppresses phosphorylation of the serine sites at positions 505, 518, 527, and / or 530 of the amino acid sequence shown in SEQ ID NO: 2, or a site on the PML protein, or the serine An agent for inducing differentiation of leukemia cells, comprising a substance that promotes dephosphorylation of a site is provided.
The present invention also relates to an agent for inducing differentiation of leukemia cells, which contains a PMLK protein expression inhibitor or function inhibitor.
The “protein expression inhibitor” in the present invention is a substance (compound) that significantly reduces protein expression or completely suppresses protein expression. The above “inhibition of expression” of the present invention includes inhibition of transcription of a gene encoding the protein and / or inhibition of translation from a transcription product of the gene.

  The PMLK in the present invention usually refers to a kinase (phosphorylating enzyme) that controls phosphorylation of PML. Specifically, it is a kinase that regulates phosphorylation of the serine site at positions 505, 518, 527, and / or 530 in the amino acid sequence shown in SEQ ID NO: 2, which is a site on the PML protein. The PMLK of the present invention includes, for example, JNK / SAPK or p38MAP kinase. Examples of the PMLK protein in the present invention include a protein having the amino acid sequence set forth in SEQ ID NO: 4, 6, or 8.

  In addition, examples of PMLK protein function-inhibiting substances in the present invention include antibodies that bind to PMLK protein (antibodies that recognize PMLK protein). The antibody is expected to inhibit the function of PMLK protein by binding to PMLK protein, and as a result, differentiation of leukemia cells is induced. The above antibody of the present invention is not particularly limited, but is preferably an antibody that does not bind to a protein other than PMLK and specifically binds to PMLK protein (recognizes PMLK protein).

  Antibodies that bind to the PMLK protein of the present invention (anti-PMLK antibodies) can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A serum is obtained by immunizing a small animal such as a rabbit with a recombinant PMLK protein expressed in a microorganism such as Escherichia coli or a partial peptide thereof as a fusion protein with natural PMLK protein or GST. This is prepared by, for example, purifying with a protein A, protein G column, an affinity column coupled with PMLK protein or a synthetic peptide, or the like. In the case of a monoclonal antibody, for example, a PMLK protein or a partial peptide thereof is immunized to a small animal such as a mouse, the spleen is removed from the mouse, and this is ground to separate the cells. Are fused using a reagent such as polyethylene glycol, and a clone producing an antibody that binds to the PMLK protein is selected from the resulting fused cells (hybridoma). Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, PMLK protein, It can be prepared by purification with an affinity column coupled with a synthetic peptide.

  The form of the antibody of the present invention is not particularly limited, so long as it binds to the PMLK protein of the present invention, in addition to the polyclonal antibody and the monoclonal antibody, a human antibody, a humanized antibody by genetic recombination, an antibody fragment thereof, Antibody modifications are also included.

  The PMLK protein of the present invention used as a sensitizing antigen for antibody acquisition is not limited to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or a human, and particularly preferably a protein derived from a human. Human-derived proteins can be obtained using the gene sequences or amino acid sequences disclosed herein.

  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of protein. As used herein, “antibody” means an antibody that reacts with a full-length protein or a partial fragment thereof.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having binding activity to proteins.

  The antibody that binds to the PMLK protein of the present invention may be used for the purpose of inducing differentiation of leukemia cells, for example. When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a human-type antibody is preferable in order to reduce immunogenicity.

Examples of the PMLK protein expression inhibiting (suppressing) substance include the following nucleic acids (a) to (c). The present invention provides an agent for inducing differentiation of leukemia cells, which comprises a compound (nucleic acid) selected from the group consisting of the following (a) to (c).
(A) An antisense nucleic acid for the transcription product of PMLK gene or a part thereof (b) A nucleic acid having ribozyme activity that specifically cleaves the transcription product of PMLK gene (c) Has an inhibitory action on the expression of PMLK gene by RNAi effect Nucleic acid

  The “nucleic acid” in the present invention means RNA or DNA. As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are several factors as described below for the action of the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing, translation initiation inhibition by hybridization with the translation initiation factor binding site, translation inhibition by hybridization with the ribosome binding site in the vicinity of the initiation codon, peptation by hybridization with the mRNA translation region and polysome binding site Elongation inhibition of de chain, and gene expression inhibition by hybrid formation at sites of interaction between nucleic acids and proteins, and the like. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Gene, Japan Biochemical) (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).

  The antisense nucleic acid used in the present invention may inhibit the expression of the PMLK gene by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the PMLK gene mRNA is designed, it is considered effective in inhibiting gene translation. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the PMLK gene is also included in the antisense nucleic acid used in the present invention. The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired animal by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the animal to be transformed, but it is not completely complementary as long as the gene expression can be effectively suppressed. May be. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is at least 15 bases, preferably 100 bases or more, more preferably 500 bases or more.

  In addition, inhibition of PMLK gene expression can also be carried out using ribozymes or DNA encoding ribozymes. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.)

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 However, it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059 .).

  Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the transcription product of the PMLK gene in the present invention using a ribozyme.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. RNAi refers to a phenomenon in which, when a double-stranded RNA having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both inhibited. Although the details of the RNAi mechanism are not clear, it is thought that the first introduced double-stranded RNA is decomposed into small pieces, and becomes an indicator of the target gene in some form, so that the target gene is decomposed. The RNA used for RNAi need not be completely identical to the PMLK gene or a partial region of the gene, but preferably has complete homology.

  In addition, if differentiation of leukemia cells is promoted by the leukemia cell differentiation inducer of the present invention, it can be applied to the treatment of leukemia. The leukemia cell differentiation inducer of the present invention is considered to be a therapeutic agent for leukemia, and this therapeutic agent is also included in the present invention. That is, the present invention provides a therapeutic agent for leukemia (pharmaceutical composition for leukemia treatment) comprising the leukemia cell differentiation inducer of the present invention as an active ingredient.

  In leukemia patients, since undifferentiated leukemia cells are proliferating, it is considered that the differentiation inducer for leukemia cells of the present invention has an action of inducing differentiation of undifferentiated cells into normal cells. It can be used for induction therapy. Therefore, the leukemia cell differentiation inducer of the present invention is very useful as a therapeutic agent for leukemia. The leukemia for which the therapeutic agent of the present invention is effective is not particularly limited, and examples thereof include acute myeloid leukemia.

  The differentiation-inducing agent or therapeutic agent for leukemia of the present invention (in this specification, “differentiation-inducing agent” and “leukemia therapeutic agent” may be collectively referred to as “drug”) is a physiologically acceptable carrier, potentiator. It can be mixed with a dosage form or a diluent and administered as a pharmaceutical composition orally or parenterally. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used. As the parenteral preparation, a dosage form such as an injection, an infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Examples of the medicine for external use include a nasal administration agent or an ointment. The preparation technique of the above dosage form so as to include the differentiation-inducing agent of the present invention which is the main component is known.

  For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant, and the like to the drug of the present invention, mixing, and compressing and shaping. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate, carboxymethyl cellulose calcium and the like are generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc, magnesium stearate and the like are known.

  The tablet containing the drug of the present invention can be coated with a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol, or the like can be used.

  An injection can be obtained by dissolving the drug of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the selection of the dispersion medium, either a water-based solvent or an oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. In the oil solvent, various vegetable oils and propylene glycol are used as a dispersion medium. At this time, a preservative such as paraben may be added as necessary. In addition, known isotonic agents such as sodium chloride and glucose can be added to the injection. Further, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

  The therapeutic agent for leukemia of the present invention can be used as an external preparation by making the drug of the present invention into a solid, liquid, or semi-solid composition. About a solid or liquid composition, it can be set as an external preparation by setting it as the composition similar to what was described previously. A semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used. A preservative such as benzalkonium chloride can be added to the composition. Also, a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.

  The necessary amount of the drug of the present invention is administered to mammals including humans within the safe dose range. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.

The present invention also provides a screening method for a therapeutic agent for leukemia (a screening method for a therapeutic agent for leukemia).
In a preferred embodiment of the screening method of the present invention, the method uses the expression level or activity of PMLK protein as an index. That is, a compound that decreases the expression or activity of PMLK protein induces differentiation of leukemia cells by activating dephosphorylation of PML protein of the protein and activating AML1 protein. Expected to have a therapeutic effect.

In the above method of the present invention, first, a test compound is brought into contact with a cell expressing the PMLK gene.
The “cell” used in the present method is not particularly limited, but is preferably a human-derived cell. As the “cell expressing the PMLK gene”, a cell expressing the endogenous PMLK gene or a cell into which an exogenous PMLK gene has been introduced and expressing the gene can be used. A cell in which an exogenous PMLK gene is expressed can usually be prepared by introducing an expression vector into which the PMLK gene has been inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  There is no restriction | limiting in particular as a test compound used for the screening method of this invention. For example, natural compounds, organic compounds, inorganic compounds, proteins, peptides and other single compounds, as well as compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts Products, plant extracts and the like, but are not limited thereto. In addition, these test compounds can be appropriately labeled as necessary. Examples of the label include a radiolabel and a fluorescent label.

  In the above method, “contact” of the test compound to the cell expressing the PMLK gene is usually performed by adding the test compound to the culture medium of the cell expressing the PMLK gene, but the method is not limited to this method. When the test compound is a protein or the like, “contact” can be performed by introducing a DNA vector expressing the protein into the cell.

In this method, the expression level or activity of PMLK protein in cells expressing the PMLK gene is then measured. The expression level can be easily measured by those skilled in the art and can be appropriately performed by a general method such as Northern blotting or Western blotting. For example, it is possible to measure using a label attached to a test compound brought into contact with a cell expressing the PMLK gene as an index.
In the present invention, “expression” means any of transcription from a gene encoding a protein (generation of mRNA) or translation from a transcription product of the gene.

Further, when the above screening method is carried out using the activity of PMLK protein as an index, for example, the phosphorylase activity of PMLK protein using PML protein as a substrate is used as an index. That is, a compound that decreases the phosphorylase activity of the PMLK protein using the PML protein as a substrate promotes differentiation induction of leukemia cells by activating the PML protein and activating the AML1 protein. It is expected to have a therapeutic effect.
Those skilled in the art can appropriately measure the activity of PMLK protein by a known method. For example, the phosphorylase activity of the PMLK protein can be appropriately measured by the method described in the examples below.

In this method, a compound that reduces the expression level or activity of the PMLK protein is then selected as compared with the case where the test compound is not contacted. The compound isolated by this method is expected to have an effect of inducing differentiation of leukemia cells and is further useful as a therapeutic agent for leukemia.
It is also possible to screen for a leukemia cell differentiation inducer from a test compound according to the method of the present invention.

  Another embodiment of the screening method of the present invention includes a method using the phosphorylation state of PML protein as an index. PML protein is activated in the dephosphorylated state. Therefore, screening for a therapeutic agent for leukemia (screening for a therapeutic agent for leukemia) is possible by detecting the phosphorylation state of PML and using the degree of phosphorylation as an index.

  In this method, first, the PML protein and a test compound are contacted. Usually, a test compound is contacted with a cell expressing the PML protein or an extract of the cell, but the test compound may be contacted with the isolated and purified PML protein.

Next, the phosphorylation state of the PML protein is measured. A person skilled in the art can appropriately measure the phosphorylation state by a general method, for example, Western blotting using a phosphorylation-specific antibody.
In the above method of the present invention, the site on the PML protein for measuring the phosphorylation state is not particularly limited, but preferably, the 505 position in the amino acid sequence of the PML protein (for example, the amino acid sequence described in SEQ ID NO: 2), Serine sites at positions 518, 527, and / or 530.
The PML protein serving as a substrate is preferably a wild-type protein that does not contain a mutation, but is a site that is phosphorylated by the PMLK protein (for example, the above-described positions 505, 518, 527, and / or 530). A partial polypeptide containing the serine site) or a mutant polypeptide containing the site.

Next, in this method, a compound that reduces the degree of phosphorylation (increases the degree of dephosphorylation) is selected as compared with the case where the test compound is not contacted. The compound thus selected is expected to have an action of inducing differentiation of leukemia cells and is useful as a therapeutic agent for leukemia.
It is also possible to screen for a leukemia cell differentiation inducer from a test compound according to the method of the present invention.

  Yet another embodiment of the screening method of the present invention is a method using the phosphorylase activity of PMLK protein as an index. That is, the method uses phosphorylation activity of PMLK protein using PML protein as a substrate as an index. A compound that decreases the phosphorylation activity of PMLK protein promotes dephosphorylation of PML protein and activates AML1 protein. As a result, differentiation of leukemia cells is induced and a therapeutic effect on leukemia is expected.

In this method, first, PML protein, PMLK protein, and a test compound are contacted. Next, the phosphorylation activity of PMLK protein using PML protein as a substrate is measured.
The PMLK protein used in the above method is preferably a wild-type protein that does not contain mutations, but a part of the amino acid sequence of PMLK protein may be substituted or deleted as long as it has an activity to phosphorylate PML protein. It may be a processed protein (polypeptide).
Further, the PML protein as a substrate is also preferably a wild-type protein that does not contain a mutation, but even if it is a partial polypeptide containing a site that is phosphorylated by the PMLK protein or a mutant polypeptide containing the site, Good.
The site on the PML protein that is phosphorylated by the PMLK protein in the above method is not particularly limited, but is preferably at position 505 in the amino acid sequence of the PML protein (for example, the amino acid sequence described in SEQ ID NO: 2). Serine sites at positions 518, 527 and / or 530.
As described above, the phosphorylating enzyme activity of the PMLK protein can be measured, for example, by Western blotting using a phosphorylation specific antibody.

  In the above method, a compound that reduces the phosphorylation activity of the PMLK protein as compared with the case where the test compound is not contacted is selected. The compound thus selected is considered to have a therapeutic effect on leukemia by inducing differentiation of leukemia cells as a result of inhibiting phosphorylation activity of PMLK protein using PML protein as a substrate.

  The compound obtained by the screening method of the present invention is expected to have an AML1 protein activation effect. Therefore, the present invention includes a method for screening for an AML1 activator comprising the steps of the above screening method.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not restrict | limited to these Examples.
[Example 1] Complex formation of AML1 and PML
FLAG was bound to AML1b, which is the full length of the AML1 gene, and AML1a, which is a splicing isoform lacking the C-terminal region of the AML1 gene. FLAG-AML1b and FLAG-AML1a were expressed in mouse myeloid progenitor cells LG by retroviral vectors. The AML1b complex was purified using an anti-FLAG antibody affinity column. As a result, many proteins were specifically included in the AML1b complex (FIG. 1A).
Subsequently, the protein with a molecular weight of 120 kDa purified together with AML1b was digested with trypsin and analyzed by mass spectrometry. As a result, four peptides were identified and found to be the amino acid sequence of the PML protein (FIG. 1B).
Further, analysis by Western blotting using an anti-PML antibody confirmed that the purified AML1b complex contained PML (FIG. 1C).
Furthermore, the K562 cell extract was immunoprecipitated using an anti-PML antibody and Western blotted with an anti-AML1 antibody. As a result, it was found that AML1 was contained in the immunoprecipitate of PML (FIG. 1D).

[Example 2] Specific binding between AML1 and PML I isoform
An expression vector of PML isoform and PML I mutant was constructed. The PML gene expresses many isoforms due to differences in splicing. Among them, PML I to VI, which are major isoforms, bind to AML1 using a mutant lacking the C-terminal or amino acids 217 to 319. (Fig. 2A).
FLAG was attached to the above PML isoform, and 293T cells were transfected with HA-AML1b by the calcium phosphate method, and after 48 hours, the cell extract was prepared. Immunoprecipitation with anti-FLAG antibody and Western blotting with anti-HA antibody showed that only PML I co-precipitated with AML1b (FIG. 2B).
Similarly, the binding of PML mutant with FLAG and AML1b was examined. Since d592 lacking the C-terminal region cannot bind to AML1b, it became clear that the C-terminal region of PML is necessary for binding to AML1b (FIG. 2C).

[Example 3] Activation of AML1-dependent transcription by PML
PML-/-and wild-type mouse embryonic fibroblasts (MEF), CCP1-luc reporter gene with AML1-binding sequence (50 ng), 200 ng pLNCX-AML1b (lanes 4-9), 250 ng pCMV-p300 (lanes 6-9), 30 ng (lanes 2, 5, 8) or 100 ng (lanes 3, 6, 9) of pLNCX-PML I and 2 ng of pRL-CMV were introduced by the calcium phosphate method. After 24 hours, luciferase activity was measured. PML significantly promoted transcription by p300 and AML1, particularly with PML-/-MEF (FIGS. 3A and B, A for PML-/-, B for wild type).

[Example 4] Promotion of differentiation of mouse myeloid cells LG into neutrophils by PML
PML I of a mutant (3R, see FIG. 2A) lacking wild type (WT) and SUMO-1 site was introduced into LG cells by LNCX retroviral vector, and expression was confirmed by Western blot. As a result, the SUML-1 PML I could not be identified in the 3R mutant (FIG. 4A).
In addition, LG cells into which LNCX-PML I, LNCX-PML I 3R, and LNCX-AML1b were introduced were cultured in the presence of G-CSF (5 ng / ml), and changes in the number of cells were examined. As a result, the expression of wild-type PML I suppressed the proliferation of LG cells similarly to AML1b, whereas PML I 3R promoted the proliferation on the contrary (FIG. 4B). Further, LG cells on day 6 of G-CSF treatment were stained with Meigimza. As a result, defoliation, which is an indicator of neutrophil differentiation, was observed in cells expressing wild type PML, but not in control cells or cells expressing PML I 3R (FIG. 4C). Similarly, LG cells on day 8 of G-CSF treatment were stained with Meigimza. As a result, segmentation was observed in control cells, but not in cells expressing PML I 3R (FIG. 4D).
Thus, wild-type PML I suppresses the proliferation of LG cells and promotes differentiation, but PML I 3R mutant acts in a dominant negative manner. It was suggested that it is important.

[Example 5] Dephosphorylation of PML accompanying M1 cell differentiation and SUMO-1 conversion Mouse myeloid progenitor cells M1 were cultured in the presence of IL-6 (20 ng / ml) for 3 to 4 days, and stained with Meigimza As a result, it was found that it was differentiated like a maurophage (FIG. 5A).
In addition, M1 cells without treatment and IL-6 treatment 3 days were immunostained with anti-PML antibody and anti-AML1 antibody. As a result, PML and AML1 colocalized in the spotted region (nuclear body) with cell differentiation (FIG. 5B).
M1 cells were treated with IL-6 for 1 to 4 days, and changes in PML were examined by Western blot. As a result, dephosphorylation of PML was induced after 2-3 days of IL-6 treatment, and SUMO-1 conversion was promoted from the 3rd day thereafter (FIG. 5C).
Thus, it was found that PML dephosphorylation and SUMO-1 formation were induced with the differentiation of M1 cells.

[Example 6] Phosphorylation site of PML
PML I was purified from an LG cell into which LNCX-FLAG-PMLI had been introduced using an anti-FLAG antibody affinity column and analyzed with a mass spectrometer after digestion with trypsin. As a result, it was shown that serine residues at positions 505, 518, 527, and 530 in the amino acid sequence of the PML I protein (SEQ ID NO: 2) are phosphorylation sites (FIG. 6A).
Mutants in which the four serine residues thus identified were substituted with alanine were prepared and introduced into 293T cells or LG cells. Analysis of mobility by Western blot confirmed that these residues were phosphorylated (FIGS. 6B and C). In addition, the mutant lacking the phosphorylation site promoted SUMO-1 formation, and in combination with these results, it was suggested that dephosphorylation is important for SUMO-1.

[Example 7] Phosphorylation of PML by PMLK proteins JNK and p38 Wild type PML I or mutant PML I 4A in which four phosphorylation sites (serines 505, 518, 527, and 530) are substituted with alanine are activated. JNK1 (lane 1, MKK7-JNK1), wild type JNK1 and active MKK7 (lane 2) or p38γ and active MKK6 were introduced into 293T cells. After 24 hours, the change in mobility was examined by Western blot. As a result, phosphorylation of PML I was induced by the activation of JNK and p38 (FIG. 7A).
293T cells introduced with HA-PML I were treated with anisomycine (10 μg / ml) that activates JNK and p38, and the change in mobility was examined by Western blot. As a result, phosphorylation of PML I was induced by anisomycine treatment (FIG. 7B).
LG cells into which HA-PML I had been introduced were treated with JNK inhibitor SP600125 (10 μM), and the change in mobility was examined by Western blot. As a result, dephosphorylation of PML I was induced by SP600125 treatment (FIG. 7C).
These results indicated that JNK and p38 phosphorylate PML.

[Example 8] Induction of cell differentiation by JNK inhibition Human myeloid leukemia cells K562 were treated with SP600125, a specific inhibitor of JNK, for 2 days, and changes in morphology were examined by Meigemsa staining. As a result, many cells lobulated like neutrophils were observed after the treatment (FIG. 8).

FIG. 1A is a photograph showing the results of purifying the AML1b complex from mouse myeloid progenitor cells LG in which FLAG-AML1b and FLAG-AML1a were expressed by a retroviral vector using an anti-FLAG antibody affinity column. FIG. 1B is a diagram showing the results of trypsin digestion of the protein having a molecular weight of 120 kDa purified together with AML1b in FIG. 1A and analysis by mass spectrometry. FIG. 1C is a photograph showing the results of Western blot analysis using an anti-PML antibody. FIG. 1D is a photograph showing the results of immunoprecipitation of K562 cell extract with anti-PML antibody and Western blotting with anti-AML1 antibody. FIG. 2A is a diagram showing the structures of PML isoforms and PML I mutants. FIG. 2B is a photograph showing the results of transfection of 293T cells with PML isoform with FLAG and HA-AML1b, immunoprecipitation with anti-FLAG antibody 48 hours later, and Western blotting with anti-HA antibody. FIG. 2C is a photograph showing the results of examining the binding of PML mutants with FLAG and AML1b by Western blotting. FIG. 3 shows that CCP1-luc reporter gene (50 ng) having AML1-binding sequence, 200 ng of pLNCX-AML1b (MEF) in PML-/-(A) and wild-type (B) mouse embryonic fibroblasts (MEF). Lanes 4-9), 250 ng pCMV-p300 (lanes 6-9), 30 ng (lanes 2, 5, 8) or 100 ng (lanes 3, 6, 9) pLNCX-PML I and 2 ng pRL It is a graph which shows the measurement result of luciferase activity 24 hours after introducing -CMV by the calcium phosphate method. FIG. 4A is a photograph showing a result of confirming expression by Western blotting after introducing PML I of a mutant (3R) lacking a wild type (WT) and SUMO-1 site into LG cells using an LNCX retroviral vector. It is. FIG. 4B is a graph showing the results of culturing LG cells into which LNCX-PML I, LNCX-PML I 3R and LNCX-AML1b have been introduced in the presence of G-CSF (5 ng / ml) and examining the change in the number of cells. It is. FIG. 4C is a photograph showing a Meigimza-stained image on day 6 of G-CSF treatment. FIG. 4D is a photograph showing a Meigimza-stained image on the 8th day after G-CSF treatment. FIG. 5A is a photograph showing the results of M1 cultured for 3-4 days in the presence of IL-6 (20 ng / ml) and stained with Meigimza. FIG. 5B is a photograph showing the result of immunostaining M1 cells untreated and IL-6 treated 3 days with anti-PML antibody and anti-AML1 antibody. FIG. 5C is a photograph showing the results of treating M1 cells with IL-6 for 1 to 4 days and examining changes in PML by Western blot. FIG. 6A shows the result of analyzing purified PML I by trypsin digestion and mass spectrometry. Serine residues at positions 505, 518, 527, and 530 in the amino acid sequence of PML I protein were shown to be phosphorylation sites. FIGS. 6B and 6C are photographs showing the results of analysis by Western blotting, in which mutants in which the four identified serine residues are substituted with alanine are prepared and introduced into 293T cells (B) or LG cells (C). is there. FIG. 7A shows that wild-type PML I (upper panel) or mutant PML I 4A (lower panel) in which four phosphorylation sites (serines 505, 518, 527, and 530) are substituted with alanine is activated JNK1 (lane 1). , MKK7-JNK1), wild-type JNK1 and active MKK7 (lane 2) or p38γ and active MKK6 together with 293T cells and 24 hours later Western blot analysis. FIG. 7B is a photograph showing the results of treating 293T cells introduced with HA-PML I with anisomycine (10 μg / ml) and examining the change in mobility by Western blot. FIG. 7C is a photograph showing the results of examining changes in mobility by Western blotting after treating L-G cells introduced with HA-PML I with SP600125 (10 μM). FIG. 8 is a photograph showing the result of K562 cells treated with SP600125 for 2 days and stained with Meigimza.

Claims (10)

  An agent for inducing differentiation of leukemia cells, comprising a PML protein expression activator or a function activator.   The agent for inducing differentiation of leukemia cells according to claim 1, wherein the functional activator of PML protein is an activator of PML protein phosphatase or an inhibitor of PML protein phosphatase.   Dephosphorylation of the serine site, which inhibits phosphorylation of the serine site at positions 505, 518, 527 and / or 530 of the amino acid sequence shown in SEQ ID NO: 2 An agent for inducing differentiation of leukemia cells, comprising a substance that promotes oxidation.   An agent for inducing differentiation of leukemia cells, comprising a PMLK protein expression inhibitor or function inhibitor. The agent for inducing differentiation of leukemia cells according to claim 4, wherein the PMLK protein expression inhibitor is a compound selected from the group consisting of the following (a) to (c).
(A) An antisense nucleic acid for the transcription product of the PMLK gene or a part thereof (b) A nucleic acid having ribozyme activity that specifically cleaves the transcription product of the PMLK gene (c) It has an inhibitory action by the RNAi effect on the expression of the PMLK gene Nucleic acid   A therapeutic agent for leukemia comprising the leukemia cell differentiation inducer according to any one of claims 1 to 5 as an active ingredient. A screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell expressing the PMLK gene (b) a step of measuring the expression level or activity of the PMLK protein in the cell (c) as compared with a case where the test compound is not contacted, A step of selecting a compound that decreases the expression level or activity A screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c).
(A) the step of contacting the PML protein and the test compound (b) the step of measuring the phosphorylation state of the PML protein (c) the compound that reduces the degree of phosphorylation compared to the case where the test compound is not contacted The process of selecting A screening method for a therapeutic agent for leukemia, comprising the following steps (a) to (c).
(A) Step of contacting PML protein, PMLK protein and test compound (b) Step of measuring phosphorylation activity of PMLK protein using PML protein as substrate (c) Compared with the case of not contacting test compound And selecting a compound that decreases the phosphorylation activity   The phosphorylation site in the PML protein is a serine site at positions 505, 518, 527, and / or 530 in the amino acid sequence of the PML protein shown in SEQ ID NO: 2. Screening method.