JPWO2014175375A1 - Method for diagnosing adult T-cell leukemia and screening method for therapeutic agent for adult T-cell leukemia - Google Patents

Abstract

A novel diagnostic method, diagnostic kit, therapeutic drug screening method, and therapeutic agent for adult T-cell leukemia are provided. The expression level of the SCYL2 gene in the cells is detected and applied to a method for diagnosing adult T cell leukemia or a method for screening therapeutic agents. Phosphorylation having an action of inhibiting phosphorylation and / or dephosphorylation of at least one site selected from the group consisting of phosphorylation sites T382, T383, and S380 of PTEN protein through regulation of SCYL2 gene expression Modulate inhibition and / or dephosphorylation.

Description

  The present invention relates to a method for diagnosing adult T-cell leukemia and a screening method for a preventive agent, progression inhibitor or therapeutic agent for adult T-cell leukemia. Furthermore, the present invention relates to a phosphorylation inhibitor or dephosphorylation agent of PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10). More specifically, the present invention relates to a method for diagnosing adult T-cell leukemia and a method for screening a therapeutic agent for adult T-cell leukemia using a gene involved in phosphorylation of PTEN that controls the PI3K / AKT signal transduction system, phosphorous It relates to oxidation inhibitors and / or dephosphorylating agents.

The PTEN gene is located at 10q23.3 on the chromosome and has been identified as a tumor suppressor (Non-patent Documents 1 and 2). PTEN protein is widely expressed in systemic cells and dephosphorylates phosphatidylinositol-3,4,5-trisphosphate (PIP3), an inositol phospholipid It is known as an enzyme that catalyzes. PIP3 is synthesized intracellularly by PI3 kinase (PI3K) and causes activation of protein kinase B (PKB) / AKT. PTEN is responsible for this dephosphorylation of PIP3 and is said to have a function of converting to phosphatidylinositol 4,5-bisphosphate (PIP2).

  Thus, PTEN negatively controls the PI3K / AKT information transmission system (Non-patent Document 3). Conversely, when PTEN activity is inhibited, PIP3 accumulates in the cell and the PI3K / AKT signaling system is activated. It has been suggested that such constant activation of the PI3K / AKT information transmission system is involved in diabetes, autism, and some cancers (Patent Document 4). In addition, it has been found that the activity is changed by modification of the PTEN molecule (Non-patent Documents 5 and 6). Maintaining the activity of PTEN is important for diagnosis and treatment of various diseases.

  Furthermore, in order to maintain the activity of PTEN, it is important to inhibit or dephosphorylate at least one site selected from the group consisting of phosphorylation sites T382, T383, and S380 of PTEN protein. It has been found by the present inventors (Patent Document 1).

International Publication No. 2010/116899

Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DH and Tavtigian SV. (1997) "Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. "Nat. Genet. 15, 356-362. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R . (1997) "PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer." Science. 275,1943-7 Li DM, Sun H. (1998) PTEN / MMAC1 / TEP1 suppresses the tumorigenicity and induces G1 cell cycle arrest in human glioblastoma cells.Proc. Nat. Acad. Sci. 95: 15406-15411 Vivanco I, Sawyers CL. (2002) The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2: 489-501. Review. Keniry M, Parsons R. (2008) The role of PTEN signaling perturbations in cancer and in targeted therapy.Oncogene 27: 5477-5485. Review. Leslie NR, Downes CP. (2004) PTEN function: how normal cells control it and tumour cells lose it.Biochem J. 2004; 382: 1-11. Review

  An object of the present invention is to provide a diagnostic method for adult T-cell leukemia.

  Another object of the present invention is to provide a screening method for a prophylactic agent, progression inhibitor, and therapeutic agent for adult T-cell leukemia.

  Still another object of the present invention is to provide a substance having phosphorylation inhibitor of PTEN and / or dephosphorylating agent or phosphorylation of PTEN.

  A further object of the present invention is to provide preventive agents, progression inhibitors and therapeutic agents for various diseases whose pathogenesis is activation of the PI3K / AKT signaling system by phosphorylation of PTEN.

  Thus, the present inventors have intensively studied and found SCYL2 as a substance involved in the inhibition of PTEN activity and the activation of the PI3K / AKT signal transduction system, thereby completing the present invention.

  The present invention relates to a method for diagnosing adult T-cell leukemia, comprising the step of determining the expression level of the SCYL2 gene in a subject-derived sample.

  In the method for diagnosing adult T-cell leukemia, the expression level of the SCYL2 gene in the subject-derived sample is increased as compared to the expression level of the SCYL2 gene of the normal control, so that the subject becomes adult T-cell leukemia. It can be shown to be affected or at risk for developing adult T-cell leukemia.

  The determination of the expression level of the SCYL2 gene can be to determine the expression level of the SCYL2 gene transcript or SCYL2 protein.

  The present invention also relates to a screening method for a preventive agent, progression inhibitor, or therapeutic agent for adult T-cell leukemia, using inhibition of SCYL2 gene or inhibition of expression of the gene as an index.

  The screening method may include the following steps. That is, a. A step of measuring the expression level of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in a cell in the presence of a test compound, and b. The expression level of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein measured in a. And a step of comparing the expression level of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in cells in the absence of the test compound.

  The present invention also relates to a diagnostic kit for adult T-cell leukemia comprising a nucleic acid or protein that binds to the SCYL2 gene, or SCYL2 protein or a peptide fragment thereof.

  In the diagnostic kit for adult T-cell leukemia, the nucleic acid that binds to the SCYL2 gene may be a combination of nucleic acids having the sequences shown in SEQ ID NO: 3 in the sequence listing and SEQ ID NO: 4 in the sequence listing.

  The present invention also relates to an inhibitor of PTEN phosphorylation and / or a dephosphorylation agent comprising a substance that inhibits the function of SCYL2.

  In the PTEN phosphorylation inhibitor and / or dephosphorylation agent, the substance that inhibits the function of SCYL2 may be an antibody against the SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of the SCYL2 gene.

  In the PTEN phosphorylation inhibition and / or dephosphorylation agent, the inhibitory nucleic acid of the SCYL2 gene may be an antisense nucleic acid, decoy nucleic acid, miRNA, shRNA or siRNA against the SCYL2 gene.

  The present invention further relates to a disease preventive agent, progression inhibitor or therapeutic agent comprising a substance that inhibits the function of SCYL2 as an active ingredient, wherein the disease is adult T cell leukemia (ATL), pancreatic cancer, autism Disease, skin cancer, malignant lymphoma, prostate cancer, endometrial cancer, cervical cancer, ovarian cancer, squamous cell carcinoma of the head and neck, esophageal cancer, bile duct cancer, prostate cancer, pheochromocytoma, penile cancer, osteosarcoma, and testis The present invention relates to a disease preventive agent, progress inhibitor or therapeutic agent selected from the group consisting of cancer.

  The substance that inhibits the function of SCYL2 may be an antibody against SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of SCYL2 gene.

  The inhibitory nucleic acid of the SCYL2 gene may be an antisense nucleic acid, decoy nucleic acid, miRNA, shRNA or siRNA against the SCYL2 gene.

  According to the present invention, a new diagnostic method for adult T-cell leukemia is provided. In addition, new screening methods for prophylactic, progression-inhibiting, or therapeutic agents for adult T-cell leukemia are provided. Furthermore, it becomes possible to suppress phosphorylation of PTEN or restore phosphorylated PTEN to an active state, prevent the onset of various diseases whose activation is one of the causes of PI3K / AKT signal transduction system, suppress progression, And a therapeutic effect can be expected. Alternatively, in the case of a phosphorylation accelerator having an opposite action thereto, it is possible to expect prevention of the onset of disease, suppression of progression, and therapeutic effect due to inactivation of the PI3K / AKT signal transduction system.

It is a figure which shows the SCYL2 gene expression level in a healthy person-derived CD4-positive T cell and an acute type ATL patient-derived T cell. It is a figure which shows the SCYL2 protein expression level in a healthy subject-derived CD4-positive T cell and an acute type ATL patient-derived T cell. It is a figure which shows the SCYL2 gene expression level in a T-ALL cell line and an ATL cell line. It is a figure which shows the SCYL2 protein expression level in a T-ALL cell line and an ATL cell line. It is a figure which shows the state of the phosphorylation of SCYL2 protein expression, PTEN protein expression, AKT protein expression, and PTEN and AKT in a T-ALL cell line and an ATL cell line. It is a figure which shows the change of the state of PTEN and AKT phosphorylation by having reduced SCYL2 gene expression in an ATL cell line. It is a figure which shows the change of the cell growth ability by reducing SCYL2 gene expression in an ATL cell line. It is a figure which shows the coupling | bonding of SCYL2 and PTEN and the coupling | bonding of SCYL2 and AKT in a cell. It is a figure which shows the state of the phosphorylation of AKT and PTEN in a SCYL2 stable expression strain and a SCYL2 expression reduction strain. It is a figure which shows the state of phosphorylation of AKT in the cell line of PTEN deficiency. It is a figure which shows the coupling | bonding of SCYL2 and PTEN in vitro. It is an in vitro kinase test, and is a figure which shows the state of PTEN phosphorylation by SCYL2. It is a figure which shows the relationship between the increase in SCYL2 expression, and the enhancement of phosphorylation of AKT and PTEN in a cancer cell line stably expressing SCYL2. It is a figure which shows the relationship between the increase in SCYL2 expression, and cell proliferation ability enhancement in the SCYL2 stable expression cancer cell line.

  In the present invention, a method for diagnosing ATL (adult T cell leukemia) and a screening method for a preventive agent, progression inhibitor or therapeutic agent for adult T cell leukemia are provided.

  ATL (Adult T-cell leukemia) refers to viral leukemia caused by infection of human T-cell leukemia virus (HTLV-1) with T cells, and HTLV-1-infected T cells become tumorous. Disease. There is a peak of onset in the 50s to 60s, progresses from subacute to chronic, progresses rapidly at the end stage, and the prognosis is poor. The origin of tumor cells is T cells, and the cuts and nuclei in the peripheral blood Leukemia cells (flower-like cells) appear, and lymph node enlargement, hepatomegaly, and spleen enlargement are frequently observed, and skin lesions are often observed.

  Here, HTLV-1 (human T-cell leukemia virus type 1) is a human retrovirus that moves into the nucleus in human T cells, which are hosts, and generates cDNA from RNA by reverse transcription. Integrate into genomic DNA.

  In the present invention, it was newly found that the SCYL2 gene or its gene product is involved in phosphorylation of PTEN. The above-described new diagnostic method and new therapeutic drug screening method are configured based on such a new discovery.

  PTEN as used herein refers to the PTEN gene or the protein encoded thereby. In vivo, the gene is located at 10q23.3 on the chromosome and has been identified as a tumor suppressor. PTEN protein refers to a protein encoded by this gene, and is known as an enzyme that catalyzes the dephosphorylation reaction of phosphatidylinositol-3,4,5-triphosphate (PIP3). In the present specification, unless otherwise specified, the term “PTEN” refers to both a full-length protein encoded by such a PTEN gene and a peptide fragment of the PTEN protein.

  The “phosphorylation site of PTEN protein” is not particularly limited, but in the context of the present invention, at least the 380th serine residue (in this specification, S380) among the amino acids constituting the PTEN protein. ), The 382 th threonine residue (also referred to herein as T382) and the 383 th threonine residue (also referred to herein as T 383), selected from the group consisting of these amino acid sites Phosphorylation at one or more sites is referred to as “PTEN phosphorylates” or “PTEN protein phosphorylates”. “Dephosphorylation” particularly means that phosphorylation of one or two sites of phosphorylation once at these residues or all the sites is not phosphorylated. Such a function can be examined by detecting the presence or absence of a dephosphorylation or phosphorylation inhibitory effect on a phosphorylated PTEN protein or a peptide fragment of the protein containing a phosphorylation site.

  In this specification, SCYL2 (SCY1-like 2) is also known as CVAK104 (Coated vesicle-associated kinase of 104 kDa) and is a protein having 929 amino acids (NCBI Reference Sequence: NP_060458.3, Sequence Listing). SEQ ID NO: 1). Has one HEAT repeat and one kinase domain. It exists in various places in the cell. For example, in the perinuclear region of the cytoplasm, the trans-Golgi retina and clathrin-coated vesicles, exist as components of AP2-containing clathrin-coated structures. It is thought to function as a Ser / Thr protein kinase.

  The SCYL2 gene in the present invention has a base sequence encoding the SCYL2 protein. Typically, it is the sequence indicated by the accession number, NM_017988.4 (SEQ ID NO: 2 in the sequence listing), but is not limited thereto.

  In the present invention, the expression “SCYL2” includes a symbol representing a gene or mRNA (GenBank accession number NM_017988.4) encoding SCYL2, and is used interchangeably for any protein, gene, or mRNA. Furthermore, in the results of analyzing base substitutions in the SCYL2 genomic region, mutations in ATL cases are also observed. These genes, proteins, and other genes, proteins, and fragments thereof containing base or amino acid mutations that can be found are also included in the definition of SCYL2 in the present invention.

  The method for diagnosing adult T cell leukemia of the present invention includes determining the expression level of the SCYL2 gene in a sample derived from a subject. Here, determining the expression level of the SCYL2 gene is not limited, but it is preferable to determine the expression level of the SCYL2 gene transcript or SCYL2 protein. Further, by comparing such expression level with the expression level of the SCYL2 gene in the normal control, if the expression level in the subject is increased relative to the normal control, the subject is suffering from adult T cell leukemia. Or it can be judged that it has the risk of such onset.

  Here, the sample derived from the subject is not limited, but is the blood or collected cells of the subject, and is particularly preferably a leukocyte derived from blood, particularly a T cell.

  Such an increase is at least 10%, preferably about 20% above normal control levels.

  Extraction and purification of a gene transcript (mRNA) or protein from a subject-derived sample can be performed according to a conventional method.

  When determining the expression level of the SCYL2 gene, the method for determining the expression level of the gene transcript is not particularly limited as long as it is a method capable of quantifying mRNA. For example, DNA chip method, Northern blot method, real-time PCR method Etc. The method for detecting mRNA here also includes detecting cDNA corresponding to mRNA.

  The method for determining the expression level of the SCYL2 protein is not particularly limited as long as the protein can be quantified. For example, there are an ELISA method using an antibody against the SCYL2 protein, a Western blotting method, an analysis method using a protein chip, and the like. It is done.

  The present invention also relates to a screening method for a preventive agent, progression inhibitor, or therapeutic agent for adult T-cell leukemia, using inhibition of SCYL2 gene or inhibition of expression of the gene as an index. In particular, inhibition of the expression of the SCYL2 gene transcript or SCYL2 protein can be used as an indicator.

Here, preferably, such a screening method comprises:
a. Measuring the amount of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in a cell in the presence of a test compound; and b. comparing the amount of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein measured in a with the amount of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in cells in the absence of the test compound.

  The cells used for such screening are not limited, but cell lines can be used. Preferably, a T cell-derived cell line is used. Such cells include, for example, HD-Mar2 cells, HPB-ALL cells, Jurkat cells, MOLT-4F cells, SKW-3 cells, TALL-1 cells, TCL-Kan cells, ILT-Mat cells, TL-Mor Cells, and MOLT-17 cells. In particular, it is desirable to use an ATL cell line.

  Alternatively, blood-derived cells obtained from subjects can be used for the screening method.

  Test compounds for screening methods include, but are not limited to, for example, low molecular weight compounds, high molecular weight compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compound live containing such compounds. Rally can be used.

  Compare the expression level of the gene when the test compound is brought into contact with the cell with the expression level of the SCYL2 gene in the absence of the candidate test compound. Therefore, it can be determined to be effective as a preventive agent, progression inhibitor or therapeutic agent for adult T-cell leukemia. Such a difference in expression level is based on the absence of the test compound as a standard, and it is indicated by a decrease of 10% or more, preferably 25% or more, more preferably 50% or more, and even more preferably 75% or more. be able to.

As a screening method for a preventive agent, progression inhibitor or therapeutic agent for adult T-cell leukemia of the present invention, a method comprising the following steps is also provided.
a. Contacting the test compound with the SCYL2 protein,
b. Detecting the binding activity of the protein and the compound, and c. Selecting a compound that binds to the protein.

  The compound that binds to the SCYL2 protein selected by the above method can inhibit the activity of the protein and can be effective in the treatment and prevention of adult T-cell leukemia.

  The method for detecting the binding activity between the test compound and the SCYL2 protein is not particularly limited as long as it can analyze the binding activity between substances, for example, Western blotting using SCYL2 antibody, surface plasmon analysis (SPR) Method and phage display.

  The test compound can be selected by selecting a compound having high binding activity on the basis of the binding constant or the like as effective for the treatment and prevention of adult T cell leukemia.

  The present invention also relates to a diagnostic kit for adult T-cell leukemia comprising a nucleic acid or protein that binds to the SCYL2 gene, SCYL2 protein or peptide fragment thereof. In particular, a nucleic acid that binds to any of the genes, or an antibody against SCYL2 or a peptide fragment thereof is preferably used, but is not limited thereto.

  The nucleic acid that binds to the SCYL2 gene is a polynucleotide that can hybridize to this gene. By using it in the DNA chip method, Northern blot method, real-time PCR method, etc., mRNA is detected and the expression level of the gene is detected. can do.

  The nucleic acid that binds to the SCYL2 gene is not limited, but may be a detection primer. The detection primer is preferably a combination of SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing, but is not limited thereto. That is, the base sequence described in SEQ ID NO: 2 in the sequence listing or a sequence complementary thereto can be used.

  In the present invention, as a preferable example of a substance having an action of inhibiting phosphorylation of PTEN and / or a substance having an action of dephosphorylating PTEN once phosphorylated, a substance which inhibits the function of SCYL2 is used. Can be mentioned. Examples of the substance that inhibits the function of SCYL2 include, but are not limited to, an antibody against SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of SCYL2 gene. For example, nucleic acid aptamers and peptide aptamer drugs that act on the SCYL2 gene or SCYL2 protein are also included.

  In the present invention, by inhibiting the function of SCYL2, it is possible to inhibit phosphorylation of PTEN and / or to dephosphorylate phosphorylated PTEN. Accordingly, it is possible to provide a phosphorylation inhibitor and / or dephosphorylation agent for PTEN, which contains a substance that inhibits the function of SCYL2 as an active ingredient. A substance having such an action is effective as a prophylactic agent, progress inhibitor or therapeutic agent for a disease whose pathogenesis is activation of the PI3K / AKT signal transduction system.

  A disease whose activation is caused by activation of the PI3K / AKT signaling system includes, but is not limited to, autism or cancer.

  Autism is a condition characterized by a qualitative disorder of interpersonal interaction, a marked abnormality in communication or its development, a markedly localized nature of activity and interest.

  Cancers include skin cancer, malignant lymphoma, prostate cancer, endometrial cancer, cervical cancer, ovarian cancer, squamous cell carcinoma of the head and neck, esophageal cancer, bile duct cancer, prostate cancer, pheochromocytoma, penile cancer, osteosarcoma, And one selected from the group consisting of testicular cancer. In addition, ATL is included.

The present invention also relates to a phosphorylating agent of PTEN, and such a drug is effective as a prophylactic, preventive, or therapeutic agent for a disease whose pathogenesis is inactivation of the PI3K / AKT signaling system. .
The SCYL2 gene or the SCYL2 protein itself is useful as such a PTEN phosphorylating agent.

  Such diseases include diabetes. Diabetes mellitus is caused by an abnormality in glucose metabolism, and exhibits a pathological condition in which the blood glucose level rises pathologically. Furthermore, an increase in blood glucose level causes various complications. PTEN gene polymorphisms are frequently observed in Japanese patients with type 2 diabetes, and the expression of PTEN increases, insulin signal is decreased and insulin resistance is caused, and may be involved in the development of type 2 diabetes. Has been suggested.

PTEN Phosphorylation Inhibition and / or Dephosphorylation Agent and Range of Therapeutic Agents PTEN Phosphorylation Inhibition and / or Dephosphorylation Agent Provided in the Present Invention, and Activity of PI3K / AKT Signaling Pathway Using This as an Active Component Prophylactic, progression-inhibiting, or therapeutic agents that have a pathogenesis as a cause include substances that inhibit the function of SCYL2. The substance that inhibits such a function includes an antibody against the SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of the SCYL2 gene. Here, the SCYL2 gene-inhibiting nucleic acid includes antisense nucleic acid, decoy nucleic acid, microRNA, shRNA, or siRNA against the SCYL2 gene.

  In the present invention, “treatment” includes improving symptoms in addition to complete healing.

<Inhibitory nucleic acid of SCYL2 gene>

  The antisense nucleic acid of SCYL2 is a single-stranded nucleic acid sequence (either RNA or DNA) that can bind to the mRNA (sense) or DNA (antisense) sequence of the SCYL2 gene. The length of the antisense nucleic acid sequence is at least about 5 nucleotides, preferably about 5-100, more preferably 9-50 nucleotides. The antisense nucleic acid of the SCYL2 gene binds to this gene sequence to form a duplex, and represses transcription or translation.

  An antisense nucleic acid can be produced using a known chemical synthesis method or biochemical synthesis method.

  An antisense nucleic acid can be introduced into a cell, for example, by various gene transfection methods such as DNA transfection or electroporation, or by using a viral vector.

  In the present invention, the decoy nucleic acid may be DNA or RNA, or may contain a modified nucleic acid within the nucleic acid. The decoy nucleic acid of the present invention can bind to a transcription factor of the SCYL2 gene and suppress promoter activity. The decoy nucleic acid of the present invention means a short nucleic acid containing a binding site for a transcription factor. By introducing this nucleic acid into a cell and binding the transcription factor to this nucleic acid, the transcription factor is bound to the original genome binding site. Binding is competitively inhibited and expression of the transcription factor is suppressed. Specifically, a decoy nucleic acid is a nucleic acid that can bind to a target binding sequence or an analog thereof.

  Examples of preferred decoy nucleic acids of the present invention include nucleic acids that can bind to transcription factors that bind to the promoter of the SCYL2 gene. The decoy nucleic acid can be designed as a single strand or a double strand including a complementary strand based on the promoter sequence of the SCYL2 gene. The length is not particularly limited, and it is a nucleic acid having at least 15 bases or 60 bases, preferably 20 to 30 bases.

  The decoy nucleic acid may be single-stranded or double-stranded, and may be circular or linear.

The decoy nucleic acid used in the present invention can be produced using a known chemical synthesis method or biochemical synthesis method. For example, a nucleic acid synthesis method using a nucleic acid synthesizer can be used.
In addition, after isolating or synthesizing a base sequence serving as a template, a PCR method or a gene amplification method using a cloning vector can also be used.

  For analysis of the transcriptional activity of the promoter when decoy nucleic acid is used, a commonly performed luciferase assay, gel shift assay, Western blotting method, FACS analysis method, RT-PCR, or the like can be employed. Commercially available kits for performing these assays can also be used.

  In the present invention, inhibitory nucleic acids can be synthetic small nucleic acid molecules that can regulate gene expression in cells by RNA interference (RNAi). Such nucleic acid molecules are, for example, siRNA, microRNA (miRNA) and shRNA molecules.

  When siRNA (small interfering RNA) molecules are used, various RNAs corresponding to the target gene can be targeted. Examples of such RNA include mRNA, mutants obtained by alternative splicing of the target gene, and post-transcriptionally modified RNA of the target gene. If alternative splicing results in a family of transcripts that are distinguished by the use of appropriate exons, siRNA molecules can be used to inhibit expression of exon portions or conserved sequences.

  The target sequence on the mRNA can be selected from a cDNA sequence corresponding to the mRNA, and is preferably a region 50 to 100 nucleotides downstream from the start codon. However, the target sequence may be located in the 5 'or 3' untranslated region or the region near the start codon.

  siRNA molecules can be designed by known methods. For example, as the target segment of the target mRNA, a continuous 15-30 base, preferably 19-25 base segment, preferably starting with AA, TA, GA or CA can be selected. The GC ratio of siRNA molecules is 30-70%, preferably 35-55%.

  siRNAs can be generated as single stranded hairpin RNA molecules that fold on their nucleic acid to generate a double stranded portion. siRNA molecules can be obtained by conventional chemical synthesis. Alternatively, siRNA molecules can be generated biologically using expression vectors containing sense and antisense siRNA sequences. In order to introduce siRNA into cells, a method of linking siRNA synthesized in vitro to plasmid DNA and introducing it into cells, a method of annealing double-stranded RNA, and the like can be employed.

  In the present invention, shRNA can also be used to cause RNA interference. shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a partial region of a single strand forms a complementary strand with another region.

  The shRNA can be designed so that part of it forms a stem-loop structure. By including a partially palindromic base sequence in a single-stranded RNA, a double-stranded structure can be formed in the molecule, resulting in a molecule of about 20 base pairs or more that becomes a hairpin structure. Such shRNA, after being introduced into the cell, is degraded to a length of about 20 bases within the cell, causing RNA interference similar to siRNA. The shRNA in the present invention is not particularly limited as long as it can cause RNAi and inhibit the function of SCYL2.

  miRNA is also an inhibitory nucleic acid based on RNA interference, and can be designed and synthesized according to shRNA or siRNA.

  PTEN phosphorylation inhibition and / or dephosphorylation agents containing these nucleic acids as active ingredients as substances capable of controlling the activity of PTEN, and treatment of diseases comprising this phosphorylation inhibition and / or dephosphorylation agents as active ingredients A drug (hereinafter referred to as “gene drug”) can be prepared. These nucleic acids are provided as they are or in a form that can be introduced into cells. A conventional method can be employed to obtain a form that can be introduced into cells. For example, the nucleic acid can be directly introduced (see US Pat. No. 5,580,859) or can be introduced in the form of a recombinant viral vector.

Examples of suitable viral vectors for cell transfer include those derived from the genome of a virus selected from the family Baculovirus, Parvoviridae, Herpesviridae, Poxviridae, Adenoviridae, Paramyxoviridae, or Picornaviridae Is used. Chimeric vectors that take advantage of the advantages of each parent vector can also be used. Such viral genomes can be replication defective, replicate according to conditions, or modified to replication competent. More specifically, when the vector is an adenovirus, for example, a replication noncompetent vector derived from the human adenovirus genome (see US Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236), adeno-associated virus, etc. Can be used. In the case of vectors derived from the retroviral genome, the main component is murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (Eg, US Pat. Nos. 6,117,681; 6,107,478; 5,658,775; 5,449,614; Buchscher (1992) J. Virol.
66: 2731-2739; Johann (1992) J. Virol. 66: 1635-1640). In addition, Sendai virus (HVJ) included in the Paramyxoviridae family can be suitably used. In this case, the polynucleotide expression vector may be introduced into the tissue or cells removed from the living body and then returned to the living body (ex vivo method). For example, an expression vector incorporating a polynucleotide is introduced into a cell by a conventional transfection method such as a microinjection method or an electroporation method.

<Antibodies to SCYL2 protein or peptide fragments thereof>

  In the present invention, it is also possible to prepare a therapeutic drug (hereinafter referred to as “antibody drug”) containing as an active ingredient a PTEN phosphorylating agent containing an antibody against the SCYL2 protein or a peptide fragment thereof as an active ingredient. A well-known method can be used for this preparation. Here, the antibody against the SCYL2 protein or a peptide thereof refers to a protein that is produced in vivo by stimulation of the SCYL2 antigen in an immune reaction and specifically binds to the SCYL2 protein or a variant thereof. A polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is particularly preferable. In addition, human antibodies, humanized antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof, such as F (ab ′) 2 and Fab fragments, and other recombinantly produced conjugates including. Further, such an antibody may be covalently bound to an enzyme such as alkaline phosphatase, horseradish peroxidase, α-galactosidase, or recombinantly fused.

  Antibodies are usually generated by immunizing a suitable animal with a peptide fragment containing the SCYL2 protein or antigen. Monoclonal antibodies include whole immunoglobulin molecules as well as Fab molecules, F (ab ') 2 fragments, Fv fragments, and other molecules that exhibit the immunological binding properties of the original monoclonal and antibody molecules. A method for producing a polyclonal antibody and a monoclonal antibody can be performed by a well-known method.

  For example, a polyclonal antibody is prepared by administering a full-length or partial fragment purified preparation of a polypeptide constituting the SCYL2 protein, a peptide having a partial amino acid sequence of the protein, and the like to an animal as an antigen. When producing antibodies, rabbits, goats, rats, mice, hamsters, etc. can be used as animals to be administered. When a peptide that is a fragment of the SCYL2 protein is used, a peptide covalently bound to a carrier protein such as mussel hemocyanin or bovine thyroglobulin can be used as the antigen. The antigen is administered 2 to 10 times every 1 to 2 weeks after the first administration. Blood is collected on days 3 to 7 after each administration, and it is confirmed by enzyme immunoassay or the like that the serum reacts with the antigen used for immunization. Serum can be obtained from a non-human mammal whose serum shows a sufficient antibody titer, and polyclonal antibodies can be separated and purified by using the serum by a well-known method.

  On the other hand, monoclonal antibodies can be prepared by, for example, using a rat whose serum has a sufficient antibody titer against SCYL2 protein or a fragment polypeptide thereof as a source of antibody-producing cells, and by fusion with myeloma cells. The hybridoma is produced. Thereafter, a hybridoma that specifically reacts with the SCYL2 protein or a fragment polypeptide thereof is selected by enzyme immunoassay or the like. From the obtained hybridoma, a monoclonal antibody having a desired property can be obtained and used as an antibody drug.

  The PTEN phosphorylation inhibition and / or dephosphorylation agent or PTEN phosphorylation agent of the present invention is provided as a parenteral or oral agent. In the case of parenteral, a gene transfer method or the like can be used effectively, but is not limited thereto.

  The preventive agent, the progression inhibitor, or the therapeutic agent for a disease comprising a substance that inhibits the function of SCYL2 of the present invention as an active ingredient is provided as a parenteral or oral agent. In the case of parenteral, a gene transfer method or the like can be used effectively, but is not limited thereto.

  In the case of parenteral administration, it may be administered to a patient alone or in combination with other agents, mixed with suitable excipients, adjuvants, and / or pharmaceutically acceptable carriers. Carriers that can be particularly preferably used include, but are not limited to, saline, buffered saline, dextrose, water, and the like. In one embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

  For parenteral administration, it can be intraarterial (eg, via the carotid artery), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal.

The preparation for oral administration can take any form of powder, granule, fine granule, dry syrup, tablet, capsule, injection, liquid and the like. Depending on the dosage form, appropriate excipients; disintegrating agents; binders; lubricants; diluents; phosphoric acid, citric acid, succinic acid, acetic acid, and Buffering agents such as organic acids or their salts; isotonic agents; preservatives; wetting agents; emulsifiers; dispersants; stabilizers; solubilizers; antioxidants such as ascorbic acid; Polypeptides (eg, less than about 10 residues) (eg, polyarginine or tripeptides); proteins (eg, serum albumin, gelatin, or immunoglobulin); hydrophilic polymers (eg, polyvinylpyrrolidone); amino acids (eg, glycine, glutamic acid) , Aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (cellulose or derivatives thereof, glucose, mannose, or dextrin) Pharmaceutical additives such as chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol); counterions (eg, sodium); and / or nonionic surfactants (eg, polysorbates, poloxamers) It can be prepared by appropriately mixing or diluting / dissolving with the product. Such substances that enhance isotonicity and chemical stability are non-toxic to recipients at the dosages and concentrations used.

  Techniques for prescribing and administration are described, for example, in the latest edition of the Japanese Pharmacopoeia and the latest supplement, “REMINGTON'S PHARMACEUTICAL SCIENCES” (Maack Publishing Co., Easton, PA).

  The phosphorylation inhibitor and / or dephosphorylation agent or disease preventive, progression inhibitor, or therapeutic agent of the present invention is a drug that is contained in an amount effective to achieve the intended purpose of the target drug. “Therapeutically effective amount” or “pharmacologically effective amount” refers to the amount of an agent that is well recognized by those of skill in the art and effective to produce a pharmacological result. The determination of a therapeutically effective dose is well known to those skilled in the art.

A therapeutically effective amount refers to the amount of a drug that reduces the disease state upon administration. The therapeutic effect and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose is preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. This dose will vary within this range depending on the mode of administration used, the sensitivity of the patient, and the route of administration. As an example, the dose of the complex is appropriately selected depending on the age and other patient conditions, the type of disease, the type of complex to be used, and the like.

  Hereinafter, the present invention will be described more specifically based on examples. However, this example shows only specific examples of the present invention, and the present invention is not limited to the following examples.

(Healthy person-derived CD4-positive cells and patient specimens)
The mononuclear cells were centrifuged using a specific gravity centrifugation method with Histopaque (Sigma Aldrich) from healthy humans who were not infected with HTLV-1 or heparinized peripheral blood of ATL patients. In acute type ATL patients, specimens with 90% abnormal peripheral blood ATL fraction were used for analysis. In addition, CD4 + T cells of healthy individuals were tested using CD4 + T cell isolation kit II (Miltenyi Biotec), and cells other than CD4 ⁺ T cells were biotin for CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ / σ, and Glycophorin A. Separation with a labeled antibody cocktail using Auto MACS (Miltenyi Biotec).

(Cell line)
The cell lines used in the experiment are as follows. HTLV-1 non-infected T cell lymphocytic leukemia (T-ALL) cell line as Jurkat, MOLT4, MKB1, KAWAI, HTLV-1 infected cell lines as HUT102, MT2, IL2 independent ATL cell lines Su9T-01, S1T, The IL2-dependent ATL cell lines KOB, KK1, and SO4 were each incubated with RPMI1640 culture medium supplemented with 10% fetal bovine serum (FBS) with or without IL-2 (50 U / ml) at 37 ° C and 5% carbon dioxide. Cultured under.

(CDNA)
First, total RNA was extracted using the triazole solution from 1 x 10 ⁷ cells cells (Invitrogen), by reverse transcription reaction by RNA PCR Kit ver3.1 AMV-RT (Takara Bio) using the total RNA 1 [mu] g CDNA was prepared. _{(MgCl 2, 10xRT buffer, dNTP} mixture, RNase inhibitor,) were mixed AMV Reverse Transcriptase and Oligo dT-Adapter primer and total RNA, was carried out once the reaction cycle (42 ° C., 30 min, 95 ° C., 5 minutes) .

[SCYL2 gene expression analysis]

1. Quantitative real time RT-PCR method
SCYL2 specific primer (forward 5′-AAGCGTGTCATTGTGCAGAG-3 ′ (SEQ ID NO: 3 in the sequence listing), reverse 5′-CCTGGATCTGAATGGAAGGA-3 ′ (SEQ ID NO: 4 in the sequence listing)), β-actin specific primer (forward 5 ′ -GACAGGATGCAGAAGGAGATTACT-3 '(SEQ ID NO: 5 in the sequence listing),
Reverse 5'-TGATCCACATCTGCTGGAAGGT-3 '(SEQ ID NO: 6 in the sequence listing)), SYBR GREEN (R) PCR Master Mix (Applied biosystems), and template cDNA are mixed, and PCR reaction is performed using ABI PRISM (R) 7000 Sequence Detection System 40 A cycle (95 degrees, 10 seconds, 60 degrees 60 seconds) was performed. The data was analyzed using the Crossing Point method for obtaining the Ct (Threshold Cycle) value from the intersection of the amplification curve and the threshold. Calculate the expression levels of SCYL2 mRNA and β-actin mRNA based on the obtained Ct value and a standard curve prepared using serial dilutions of standard samples (cDNA derived from Jurkat cells). Expression level / β-actin gene expression level was calculated and corrected by dividing by the value of the reference sample.

2. SCYL2 antibody and other antibodies

Uzaki anti-human SCYL2 antibody consists of three SCYL2 peptides (amino acid residues TDNTKRNLTNGLNA 762-775 (SEQ ID NO: 7 in the sequence listing), QLSQQKPNQWLNQFV 855-869 (SEQ ID NO: 8 in the sequence listing), and CTTMTNSSSASNDLKDLFG 912-929 (sequence listing). SEQ ID NO: 9)) was synthesized, rabbits were immunized and serum was used. Other antibodies include mouse anti-β-actin (AC-74) monoclonal antibody (A5316), rabbit anti-Phospho-PTEN (Ser380 / Thr382 / Thr383) polyclonal antibody (# 9554), rabbit anti-PTEN polyclonal antibody (# 9559), rabbit Anti-phospho-AKT (Ser473) polyclonal antibody (# 9271) and rabbit anti-AKT polyclonal antibody (# 9272) (Cell Signaling TECHNOLOGY) were used. Further, horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody (NA931V) (GE Healthcare) and HRP-labeled anti-IgG antibody (P0399) (Dako Cytomation) were used as secondary antibodies.

3. Western blotting Cells 1 x 10 ⁶ cells after the recovery, phosphate-buffered saline (10 mM phosphate buffer solution, 120 mM NaCl, 2.7 mM KCl , pH 7.6) (PBS) and washed with buffer, 150 [mu] l of 1x SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, 0.01% bromophenol blue) was added to lyse the cells. After boiling at 95 ° C. for 5 minutes, the sample was subjected to 10% polyacrylamide gel (acrylamide: bisacrylamide = 36.5: 1) electrophoresis. Electrophoresis was performed with a miniprotean 3 cell (Bio-Rad) at a constant voltage of 100 V for 2 hours. The gel was transferred to a PVDF membrane (Millipore) using a mini-trans blot cell (Bio-Rad) (400 mA, 3 hours). The transfer membrane was tris buffered saline / Tween 20 supplemented with 1% bovine serum albumin (BSA). A blocking reaction was performed with (TBS / T) buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1% Tween20) at room temperature for 2 hours. The primary antibody solution diluted 2000 times with Can Get signal (R) Solution 1 (TOYOBO) was reacted overnight at 4 degrees and washed with TBS / T buffer for 5 minutes x 3 times. Furthermore, Can Get signal (R)
The solution was reacted with a secondary antibody solution diluted 3000 times with Solution 2 (TOYOBO) for 1 hour at room temperature, and then washed with TBS / T buffer for 5 minutes × 3 times. After washing, chemiluminescence was performed using a Lumi lightPLUS Western Blotting kit (Roche Applied Science), and image analysis was performed using a lumino image analyzer LAS-3000 (Fujifilm).

4). SCYL2 gene expression analysis
SCYL2 mRNA expression by quantitative real time RT-PCR, CD4 positive T cells derived from healthy subjects, and T cells derived from acute ATL patients, 4 T-ALL cell lines (Jurkat, MOLT4, MKB-1, KAWAI), Quantification was performed using HTLV-1 + cell lines (HUT102, MT2) and ATL cell lines (ED-40515 (−), Su9T-01, S1T, KOB, KK1, SO4). Similarly, SCYL2 protein expression was quantified by Western blotting for the same cells. As a result, an increase in SCYL2 mRNA expression (FIG. 1) and an increase in SCYL2 protein expression (FIG. 2) were observed in T cells derived from acute type ATL patients as compared to CD4 positive T cells derived from healthy subjects as controls. Furthermore, the ATL cell line tended to slightly increase the expression level compared to 4 T-ALL cell lines (FIG. 3 for SCYL2 mRNA expression and FIG. 4 for SCYL2 protein expression). In the figure, p-PTEN (STT) represents p-PTEN (Ser380 / Thr382 / Thr383).

[SCYL2 expression and AKT / PTEN phosphorylation status analysis]

1.Antibody

The same antibody as in Example 2 was used.

2. Phosphorylation of AKT and PTEN In the same manner as in Example 1, the expression of PTEN and AKT proteins was analyzed by Western blotting using CD4 positive T cells derived from healthy subjects, ATL cells derived from acute ATL patients, and ATL cell lines. . Although AKT and PTEN protein expression was not changed in ATL patients derived from acute type ATL patients compared to CD4 positive T cells derived from healthy subjects as controls, phosphorylated AKT (Ser473) and phosphorylated PTEN ( Increased Ser380 / Thr382 / Thr383) was observed (FIG. 2).

  Compared to 4 T-ALL cell lines, 8 ATL cell lines did not show changes in AKT and PTEN protein expression, but increased phosphorylation (FIG. 5). This suggested that AKT and PTEN phosphorylation was enhanced by high expression of SCYL2.

[SCYL2 gene expression reduction experiment]

1.Antibody

The same antibody as in Example 2 was used.

2. Preparation of SCYL2 gene expression modified vector
As a forward primer to amplify the SCYL2 coding region, (EcoRI-SCYL2 5'-GGAATTCGATGGAGTCCATGCTTAATAAATTGAAG-3 ') (SEQ ID NO: 10 in the sequence listing) and as a reverse primer (XbaI-SCYL2 5'-GCTCTAGATCACCCAAAAAGATCTTTTAAATCATTG-3') ( Sequence number 11) of the sequence listing was used. For amplification, Prime star MAX DNA polymerase kit (TaKaRa) was used and PCR40 reaction cycle (98 ° C, 10 seconds, 60 ° C, 10 seconds, 72 ° C, 2 minutes) was performed. The obtained PCR product was inserted into the EcoRI and XbaI sites of pCMV26 (SIGMA) to prepare an SCYL2 gene expression increasing vector (pCMV26-SCYL2).

  Next, sh-SCYL2-sence (5'-gatccGCCCGTTAATACAAACCAGTTCAAGAGACTGGTTTGTATTAACGGGCTTTTTTT-3 ') (sequence number 12 in the sequence listing) and sh-SCYL2-antisence (5'-aattAAAAAAAGCCCGTTAATACAAACCAGTCTCTTGAACTGGTTTG'out sequence sequence TMTTATTAACGGGCg-3) An SCYL2 expression-reducing vector was prepared using the RNAi system (Clontech) (sh-SCYL2).

3. Gene transfer
2 μg of the expression vector was introduced into the 293T cell 6-well plate with the gene introduction reagent HilyMax (DOJINDO) and cultured for 2 days. 2 μg of expression vector was introduced into ATL cell line KK1 cell 2 × 10 ⁶ cells with Amaxa Nucleofector (Amaxa) and cultured for 2 days.

4). Cell proliferation experiment
2 × 10 ³ cells were cultured in a 96-well plate, cell counting kit-8 (DOJINDO) was added at a predetermined time, and absorbance at 490 nm was measured to analyze cell proliferation ability.

  By using the above method, the SCYL2 gene of ATL cell line KK1, which is highly expressed in SCYL2 gene and enhanced phospho-AKT (Ser473) and Phospho-PTEN (Ser380 / Thr382 / Thr383), is expressed using sh-SCYL2. The AKT / PTEN phosphorylation state and cell proliferation ability at that time were examined.

After introducing sh-SCYL2 with Amaxa Nucleofector (Amaxa) and culturing for 2 days, the change in phosphorylation state of AKT and PTEN was confirmed by Western blotting, and cell proliferation ability was added with cell counting kit-8 (DOJINDO), Absorbance at 490 nm was measured and analyzed.
sh-SCYL2 significantly reduced SCYL2 expression and did not change AKT and PTEN protein expression, but suppressed phospho-AKT (Ser473) and Phospho-PTEN (Ser380 / Thr382 / Thr383) ( FIG. 6). Furthermore, a decrease in cell proliferation ability was also observed (FIG. 7).

  From the above, suppression of ACY cell proliferation was demonstrated through suppression of AKT and PTEN phosphorylation by suppressing the expression of SCYL2.

[SCYL2-PTEN or SCYL2-AKT binding experiment]
1． Immunoprecipitation method Cells were collected, washed with PBS buffer, and 1% NP-40 buffer was added to lyse the cells. After centrifugation, 1-2 μg of antibody was added to 100-200 μg of cell lysate and reacted at 4 ° C. overnight. 50 μl of 50% slurry Protein G Sepharose ™ 4 fast flow (GE Healthcare) was added and reacted at 4 ° C. for 2 hours. Protein G Sepharose TM4 fast flow was washed 3 times with PBS, SDS sample buffer was added, boiled at 95 ° C. for 5 minutes, and Western blot analysis was performed.

2. Purification of Flag fusion protein
The gene was inserted into pCMV26 (SIGMA). The vector was introduced into the 293T cell line, and 2 days later, 1% NP-40 buffer was added to lyse the cells. 50% slurry Anti-Flag M2 affinity Gel (SIGMA) was added and reacted at 4 ° C. overnight. The Flag fusion protein was eluted with 3xFlag peptide (SIGMA).

The binding of SCYL2 and PTEN was analyzed by immunoprecipitation experiment.
Endogenous PTEN of 293T cells and KK1 cells was immunoprecipitated with PTEN antibody, and Western blotting was performed with SCYL2 antibody.

As a result, it was confirmed that SCYL2 was present in the PTEN immunoprecipitate for both cells (FIG. 8).
Furthermore, using the Flag-SCYL2 / 293T stable expression strain, immunoprecipitation was performed with Flag antibody (mouse anti-Flag M2 monoclonal antibody (F1804) (SIGMA ALDRICH)), and Western blotting was performed with AKT and PTEN antibodies. The presence of AKT and PTEN was observed in the Flag (SCYL2) immunoprecipitate (FIG. 8).
From the above, it was revealed that SCYL2-PTEN and AKT are bound intracellularly.

[Detection of AKT-PTEN phosphorylation status]
AKT phosphorylation state and binding when SCYL2 was overexpressed in PC3 cell line lacking PTEN were analyzed by Western blotting and immunoprecipitation.
When Flag-SCYL2 was expressed in the PC3 cell line, no change in AKT protein expression was observed, and no change in phospho-AKT (Ser473) was observed (FIG. 10). On the other hand, when PTEN was expressed, a decrease in phospho-AKT (Ser473) was observed, and when SCYL2 and PTEN were expressed simultaneously, an increase in phospho-AKT (Ser473) was observed compared to PTEN alone. Furthermore, SCYL2 cannot bind to AKT in the absence of PTEN, but it was revealed that SCYL2 binds to PTEN and AKT in the presence of PTEN (FIG. 9).
This suggests that SCYL2 regulates AKT phosphorylation through binding to PTEN.

[In vitro SCYL2-PTEN binding experiment]
1. Purification of GST (glutathione S-transferase) fusion protein
The gene was inserted into the pGEX-6P-1 vector. The vector was transformed into E. coli BL21DE3 and cultured at 37 ° C. The fusion protein was expressed by adding 1 mM IPTG at 30 ° C. for 3 hours. 1% NP-40 buffer was added to E. coli, and ultrasonic grinding was performed. After centrifugation, the supernatant was collected, 50% slurry Glutathione Sepharose TM4 4B (GE Healthcare) was added, and the mixture was reacted at 4 ° C. overnight. The GST fusion protein was eluted with an elution buffer (20 mM reduced glutathione, 100 nMTris-HCl (pH 8.0), 120 mM NaCl).

  It was confirmed by in vitro binding test whether SCYL2 and PTEN were directly bound. GST-SCYL2 purified from E. coli and 293T cell lysate were mixed and allowed to react overnight at 4 ° C. Add 50% slurry Glutathione Sepharose TM4 4B (GE Healthcare), react at 4 ° C for 2 hours, wash 3 times with PBS, add SDS sample buffer, boil at 95 ° C for 5 minutes, Western blot with PTEN antibody Analysis was performed. As a result, it was revealed that GST-SCYL2 and PTEN were bound (FIG. 11).

In vitro binding test (Pull-down)
The purified GST-SCYL2 protein and 293T cell lysate were mixed and reacted at 4 ° C. overnight. Add 50% slurry Glutathione Sepharose TM4 4B (GE Healthcare), react at 4 ° C for 2 hours, wash 3 times with PBS, add SDS sample buffer, boil at 95 ° C for 5 minutes, Western blot with PTEN antibody Analysis was performed.

[In vitro kinase test]
GST-PTEN protein, Flag-SCYL2 protein and 100 μM ATP were added to a kinase solution (25 mM Tris-HCl (Ph8.0), 25 mM β-glycerophosphate, 25 mM MgCl ₂ ) and reacted at 37 ° C. for 30 minutes. Western blotting was performed with PTEN phosphorylated antibody.

  We examined whether PTEN phosphorylation was caused by SCYL2 in an in vitro kinase test.

  When Flag-SCYL2 purified from 293T cell line and GST-PTEN purified from E. coli were mixed in the presence of ATP, enhancement of Phospho-PTEN (Ser380 / Thr382 / Thr383) was observed (FIG. 12).

  This suggests that SCYL2 acts as a kinase using PTEN as a substrate.

[Changes in AKT and PTEN phosphorylation status in solid cancer cells]
1. Production of stable expression cell lines
After gene introduction into the 293T cell line and U2OS cell line, G418 (GIBCO) was added to the culture medium at a concentration of 500 μg / mi, and drug selection was performed to obtain a stable expression cell line.

  In the stable expression strain of Flag-SCYL2 / U2OS, enhancement of phospho-AKT (Ser473) and Phospho-PTEN (Ser380 / Thr382 / Thr383) was observed as SCYL2 expression increased (FIG. 13). Furthermore, enhancement of cell proliferation ability was also observed (FIG. 14).

  The results of these experiments revealed that SCYL2 is highly expressed in ATL cells. In addition, it was shown to act as a kinase that directly binds to PTEN and phosphorylates Ser380 / Thr382 / Thr383 sites, and phosphorylates PTEN to enhance AKT phosphorylation and cell proliferation ability. From the above, we found the possibility of SCYL2 as a therapeutic target.

Claims (13)

  A method for diagnosing adult T-cell leukemia, comprising a step of determining the expression level of SCYL2 gene in a sample derived from a subject.   When the expression level of the SCYL2 gene in the sample derived from the subject is increased compared to the expression level of the SCYL2 gene of the normal control, the subject is suffering from adult T-cell leukemia or adult T-cell leukemia The method for diagnosing adult T-cell leukemia according to claim 1, which is shown to have a risk of onset.   The diagnostic method according to claim 1 or 2, wherein the determination of the expression level of the SCYL2 gene is to determine the expression level of a SCYL2 gene transcript or SCYL2 protein.   A screening method for a preventive agent, progression inhibitor or therapeutic agent for adult T-cell leukemia using SCYL2 gene inhibition or gene expression inhibition as an index. The method for screening a prophylactic, progression-inhibiting, or therapeutic agent for adult T-cell leukemia according to claim 4, comprising the following steps:
measuring the expression level of SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in a cell in the presence of a test compound; and
b. comparing the expression level of the SCYL2 gene, SCYL2 gene transcript or SCYL2 protein measured in a with the expression level of the SCYL2 gene, SCYL2 gene transcript or SCYL2 protein in the cell in the absence of the test compound.   A diagnostic kit for adult T-cell leukemia comprising a nucleic acid or protein that binds to the SCYL2 gene, or SCYL2 protein or a peptide fragment thereof.   The diagnostic kit for adult T-cell leukemia according to claim 6, wherein the nucleic acid binding to the SCYL2 gene is a combination of nucleic acids having the sequences shown in SEQ ID NO: 3 in the sequence listing and SEQ ID NO: 4 in the sequence listing.   An inhibitor of PTEN phosphorylation and / or a dephosphorylation agent comprising a substance that inhibits the function of SCYL2.   The phosphorylation inhibitor and / or dephosphorylation agent for PTEN according to claim 8, wherein the substance that inhibits the function of SCYL2 is an antibody against SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of SCYL2 gene.   The PTEN phosphorylation inhibition and / or dephosphorylation agent according to claim 9, wherein the inhibitory nucleic acid of the SCYL2 gene is an antisense nucleic acid, decoy nucleic acid, miRNA, shRNA or siRNA against the SCYL2 gene.   A prophylactic agent, progression inhibitor or therapeutic agent for a disease comprising a substance that inhibits the function of SCYL2 as an active ingredient, wherein the disease is adult T cell leukemia (ATL), pancreatic cancer, autism, skin cancer, From the group consisting of malignant lymphoma, prostate cancer, endometrial cancer, cervical cancer, ovarian cancer, squamous cell carcinoma of the head and neck, esophageal cancer, bile duct cancer, prostate cancer, pheochromocytoma, penile cancer, osteosarcoma, and testicular cancer A prophylactic agent, progress inhibitor or therapeutic agent for a disease, which is one type selected.   The disease preventive agent, progress inhibitor or therapeutic agent according to claim 11, wherein the substance that inhibits the function of SCYL2 is an antibody against SCYL2 protein or a peptide fragment thereof, or an inhibitory nucleic acid of SCYL2 gene. The PTEN phosphorylation inhibition and / or dephosphorylation agent according to claim 12, wherein the inhibitory nucleic acid of the SCYL2 gene is an antisense nucleic acid, decoy nucleic acid, miRNA, shRNA or siRNA against the SCYL2 gene.

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