WO2019088164A1 - Novel polynucleotide and utilization thereof - Google Patents

Novel polynucleotide and utilization thereof Download PDF

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WO2019088164A1
WO2019088164A1 PCT/JP2018/040494 JP2018040494W WO2019088164A1 WO 2019088164 A1 WO2019088164 A1 WO 2019088164A1 JP 2018040494 W JP2018040494 W JP 2018040494W WO 2019088164 A1 WO2019088164 A1 WO 2019088164A1
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polynucleotide
polypeptide
rarb
cells
tbl1xr1
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PCT/JP2018/040494
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French (fr)
Japanese (ja)
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元博 加藤
朋生 大隅
信敬 清河
一彦 中林
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国立研究開発法人国立成育医療研究センター
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to novel polynucleotides related to acute promyelocytic leukemia, and uses thereof.
  • APL Acute promyelocytic leukemia constitutes 5-10% of acute myeloid leukemia (AML).
  • AML is defined by unique morphological features and is typically associated with abnormal promyelocytes, which contain azulotic granules.
  • the genomic root of APL is characterized by a reciprocal translocation t (15; 17) (q22; q21) that causes a fusion between the promyelocytic leukemia gene (PML gene) and the retinoic acid receptor alpha (RARA) gene (Non-Patent Documents 1 to 3).
  • APL cells having PML-RARA respond well to all-trans retinoic acid (ATRA; synonymous with all-trans retinoic acid) and arsenic trioxide (ATO; synonymous with arsenite) (Non-patent document 4) 6) Recent clinical trials have achieved excellent results for typical APL with PML-RARA.
  • ATRA all-trans retinoic acid
  • ATO arsenic trioxide
  • APL cases lack PML-RARA, many have reciprocal translocations between RARA and other partners (such as PLZF, NPM, NUMA1, or TBL1XR1). After all, many of the known translocations associated with APL are associated with RARA.
  • One aspect of the present invention aims to provide novel polynucleotides related to APL, and uses thereof.
  • RARB retinoic acid receptor beta
  • the present invention comprises any one of the following modes.
  • novel polynucleotides related to APL can be provided.
  • it can be used for elucidation of the onset mechanism of APL in which RARA is considered not to be involved, development of a therapeutic method or a therapeutic agent thereof, and the like.
  • FIG. 1 shows morphological, cytogenetic and genomic characteristics of APL cases (UPN1) without RARA translocation.
  • FIG. 5 shows the influence of TBL1XR1-RARB on in vitro transcriptional activity, differentiation and homodimerization.
  • FIG. 5 shows the influence of TBL1XR1-RARB on differentiation and proliferation ex vivo.
  • FIG. 5 shows the effects of various retinoid additions on PML-RARA and TBL1XR1-RARB.
  • protein can also be referred to as "polypeptide”.
  • the “protein” includes a structure formed by peptide bonding of amino acids, but may further include a structure such as a sugar chain or an isoprenoid group.
  • Protein includes polypeptides containing known analogues of naturally occurring amino acids that can function similarly to naturally occurring amino acids.
  • nucleic acid includes polynucleotides consisting of any simple nucleotides and / or modified nucleotides, such as cDNA, mRNA, total RNA, hnRNA and the like.
  • Modified nucleotides include inosine, acetyl cytidine, methyl cytidine, methyl adenosine, and phosphate esters including methyl guanosine, as well as nucleotides that can be generated by the action of ultraviolet light and chemicals.
  • polynucleotide means a polymer of nucleotides.
  • gene includes not only double-stranded DNA but also single-stranded DNA and RNA (such as mRNA) such as the sense strand and antisense strand that constitute it.
  • DNA includes, for example, cDNA and genomic DNA as obtained by cloning, chemical synthesis techniques, or a combination thereof. That is, the DNA may be a "genomic” form DNA including non-coding sequences such as introns which are contained in the genome of animals etc., or may be obtained through mRNA using reverse transcriptase or polymerase. Or a "transcribed" form of DNA that does not contain non-coding sequences such as introns.
  • APL Acute Promyelocytic Leukemia
  • APL is a kind of acute myeloid leukemia, and is a generic term for leukemia in which promyelocytes become cancerous.
  • Acute promyelocytic leukemia is also referred to as acute promyelocytic leukemia.
  • “healthy person” refers to a normal individual who is found to be free of any disease, including APL.
  • leukemia non-affected person refers to an individual who is at least found to be in a state not suffering from leukemia (including APL), and "APL non-affected person” is at least suffering from APL. Refers to an individual who is found to be absent.
  • ⁇ having the risk (risk) of onset is intended to mean that the possibility of onset is higher than that of a healthy person, although it does not lead to onset, and has a risk of onset of APL. That includes the situation before APL and likely to reach APL.
  • test refers to a subject in a subject of a test (sometimes referred to as a "subject”, preferably a human being) that does not require identification (diagnosis) by a doctor.
  • a subject sometimes referred to as a "subject", preferably a human being
  • the obtained test result can be one of the materials for diagnosis made by a doctor.
  • examining the presence or absence of the predisposing factor of APL is a concept including examining the possibility of developing APL in the future.
  • examining the presence or absence of the onset of APL refers to examining whether or not the onset of APL has occurred.
  • treatment includes completely curing or reducing the symptoms of the target disease, suppressing the aggravation of the symptoms of the target disease, suppressing or delaying the onset of the target disease. That is, it includes “prevention” when the subject does not develop the target disease.
  • polynucleotide according to one embodiment of the present invention
  • this polynucleotide is a polynucleotide described in any of the following (1) to (5).
  • the polypeptide represented by the amino acid sequence of SEQ ID NO: 1 is one of the polypeptides obtained in the examples described later.
  • This sequence identity is preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, particularly preferably 95% or more, 96% It is particularly preferable that the content is 97% or more, 98% or more, or 99% or more.
  • the amino acid sequence of SEQ ID NO: 1 has an amino acid sequence in which 1 to 100 amino acids have been substituted, deleted, inserted and / or added, and functions the same as the polypeptide having the amino acid sequence of SEQ ID NO: 1 It may be a polynucleotide encoding a polypeptide which it has.
  • the number of amino acids substituted, deleted, inserted and / or added is preferably 1 to 54, more preferably 1 to 27, and 1 to 20. More preferably, 1 to 15, 1 to 10 or 1 to 6 is particularly preferable.
  • polynucleotide consisting of a sequence complementary to the polynucleotide described in (3) above, and has the same function as a polypeptide having the amino acid sequence of SEQ ID NO: 1 It may be a polynucleotide encoding a polypeptide.
  • This polynucleotide preferably has a sequence identity of 85% or more to the nucleotide sequence of the polynucleotide described in (3) above.
  • the sequence identity is preferably 88% or more, more preferably 90% or more, still more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is particularly preferable to be the above.
  • the above-mentioned stringent conditions include, for example, the conditions described in the reference document [Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)]. More specifically, under stringent conditions, for example, 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 Conditions for hybridization by incubating a solution containing ⁇ Denhardt and 100 mg / mL herring sperm DNA at 65 ° C. for 8 to 16 hours with a probe may be mentioned.
  • the fusion polypeptide of a part or the entire length of RARB and a part or the entire length of TBL1XR1 described in (1) to (4) above includes, for example, a part (the N-terminal side) of the modulating region of RARB. Is replaced with a TBL1XR1 N-terminal fragment containing a LisH domain, or a part (N-terminal part) of the modulating region of RARB is a TBL1XR1 N containing a LisH domain and an F-box-like domain. Those which have been replaced by fragments on the terminal side, and the like can be mentioned.
  • one of the polypeptides obtained in the examples described later is also a novel polypeptide produced by translocation within one gene or mutual translocation between two genes.
  • the method for obtaining (isolating) the polynucleotide according to one embodiment of the present invention is not particularly limited, and, for example, a probe that specifically hybridizes with a part of the base sequence of the above-mentioned polynucleotide It may be prepared and screened for genomic DNA library or cDNA library. Alternatively, the polynucleotide according to the present invention may be synthesized according to a nucleic acid synthesis method such as the phosphoroamidite method.
  • a method for obtaining the polynucleotide according to an embodiment of the present invention a method using a nucleic acid amplification method such as PCR can be mentioned.
  • a primer is prepared from each of the 5 'and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and these primers are used for PCR using genomic DNA or cDNA as a template. And amplify the DNA region sandwiched between the two primers. Thereby, a large amount of DNA fragments containing the polynucleotide according to one embodiment of the present invention can be obtained.
  • the polynucleotide for example, DNA
  • the type of vector may be an autonomously replicating vector such as a plasmid, or it may be integrated into the host cell's genome when introduced into the host cell and replicated together with the host cell's chromosomes. Good.
  • the vector is preferably an expression vector.
  • a polynucleotide according to an embodiment of the present invention is functionally linked to elements necessary for transcription, such as, for example, a promoter sequence.
  • a promoter sequence is a DNA sequence that shows transcriptional activity in a host cell. The type of promoter sequence to be used may be appropriately selected according to the type of host cell.
  • the polynucleotide according to one embodiment of the present invention may be functionally linked to an appropriate terminator, as needed.
  • An appropriate type of terminator may be appropriately selected depending on the type of host cell.
  • the recombinant vector according to one embodiment of the present invention may further have an element such as a polyadenylation signal, a transcription enhancer sequence, or a translation enhancer sequence.
  • the recombinant vector according to one embodiment of the present invention may further have a DNA sequence that allows the vector to replicate in a host cell.
  • the recombinant vector according to one embodiment of the present invention may further have a selectable marker.
  • a selectable marker can include, for example, a drug resistant gene to a drug such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin or hygromycin.
  • the polypeptide according to an embodiment of the present invention is a polypeptide encoded by any of the polynucleotides according to an embodiment of the present invention described above.
  • the polypeptide having the amino acid sequence of SEQ ID NO: 1 is the polypeptide obtained in the example.
  • the polypeptide forms, for example, a homodimer and suppresses the activation of the retinoic acid pathway by ATRA administration, and the above-mentioned cells (especially, blood cells and cells of immune system including promyelocytes). It is presumed to have the function of suppressing differentiation.
  • the origin of the polypeptide is not limited, and may be isolated from natural sources, chemically synthesized, or produced using genetic engineering techniques. More specifically, the polypeptide includes, in its category, an isolated and purified natural polypeptide, a chemically synthesized polypeptide, and a polypeptide produced from a host cell based on genetic engineering technology. The host cell will be described in detail in the section describing “transformant”.
  • polynucleotide and polypeptide according to the embodiment of the present invention described above are presumed to be one of the causative gene and protein of APL, for example, elucidation of the onset mechanism of APL, therapy for APL (therapeutic agent Development of APL, diagnosis of APL, and selection of therapy for APL.
  • a transformant can be produced by introduceably expressing a polynucleotide or a recombinant vector according to one embodiment of the present invention into a suitable host cell.
  • the polynucleotide and the recombinant vector according to one embodiment of the present invention are generically referred to as "foreign nucleic acid molecule".
  • the method for introducing the foreign nucleic acid molecule into the host cell may be selected according to the type of host cell, as exemplified below. Also, the method of obtaining the offspring of the transformant according to one embodiment of the present invention may be selected according to the type of transformant.
  • Host cells include, for example, bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells.
  • Higher eukaryotic cells include, for example, plant cells and animal cells.
  • Animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, mammalian cells and the like.
  • bacterial cells examples include gram positive bacteria such as Bacillus or Streptomyces; Gram negative bacteria such as E. coli. Transformation of these bacterial cells may be performed by, for example, a protoplast method or a method using competent cells.
  • yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces.
  • methods for introducing foreign nucleic acid molecules into a yeast host include electroporation, spheroplast method, lithium acetate method and the like.
  • filamentous fungal cells examples include filamentous fungal cells.
  • transformation can be carried out by incorporating foreign nucleic acid molecules into the host chromosome to obtain recombinant host cells. Integration of foreign nucleic acid molecules into the host chromosome can be performed, for example, by homologous recombination or heterologous recombination.
  • insect cells include, for example, silkworm cells.
  • foreign nucleic acid molecules and baculovirus are co-introduced into insect cells to obtain recombinant baculovirus in the culture supernatant of insect cells.
  • the insect cells are then infected with the recombinant baculovirus.
  • a method of co-introduction for example, calcium phosphate method or lipofection method can be mentioned.
  • mammalian cells examples include HEK 293 cells, HeLa cells, COS cells, BHK cells, CHL cells, CHO cells and the like.
  • the cells used be blood cells or cells of the immune system. Blood cells and cells of the immune system, for example, B cell lines such as Jeko-1 cells, Raji cells, Ramos cells etc .; T cells such as Jurkat cells, MOLT-4 cells, TG40 cells, U-937 cells etc.
  • basophil cell lines such as KU812-F cells, RBL-2H3 cells
  • granulocyte cell lines such as HMC-1 cells, Ku812 cells, RBL cells
  • lymphoblasts such as MCL-5 cells, MOLT-4 cells Cell lines
  • Neutrophil-like cell lines such as NB-4 cells, THP-1 cells
  • Macrophage cell lines such as 416 B cells, MOLM-14 cells, THP-1 cells
  • Leukocytes such as BJAB cells, CML cells, Daudi cells Cell line
  • Lymphoma cell line such as A20 cell, BC-3 cell, CA46 cell, HCT116 cell, ST-486 cell, etc .
  • Lymphoblast such as ESB cell, TCLB cell Tumor cell lines
  • leukemia cell lines such as HL-60 cells and K562 cells
  • the blood cells used in the present invention may be differentiated or undifferentiated.
  • undifferentiated cells examples include, for example, hematopoietic progenitor cells, hematopoietic stem cells and the like.
  • undifferentiated hematopoietic cells artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), blood cells (such as cord blood cells), cells obtained by reprogramming differentiated cells, and the like can be exemplified.
  • iPS cells pluripotent stem cells
  • ES cells embryonic stem cells
  • blood cells such as cord blood cells
  • cells obtained by reprogramming differentiated cells and the like
  • transformation of mammalian cells for example, electroporation, calcium phosphate method, lipofection method and the like can be used.
  • an electroporation method for example, an electroporation method, an Agrobacterium method, a particle gun method or the like can be used.
  • the transformant described above is cultured or grown under conditions that allow expression of the introduced foreign nucleic acid molecule. Then, if necessary, the polypeptide according to one embodiment of the present invention is isolated and purified from the transformant. Alternatively, if necessary, the transformant itself is used for various evaluations and the like.
  • transformant is not limited to cells. That is, transformants may be, for example, tissues, organs, and individuals transformed with foreign nucleic acid molecules. However, it may be preferable that the non-cell transformant is of non-human origin, and in particular, the individual is preferably of non-human origin.
  • kits In an example of a kit according to an embodiment of the present invention, 1) a polynucleotide according to an embodiment of the present invention, 2) a polypeptide according to an embodiment of the present invention, and 3) an embodiment of the present invention And at least one selected from such cells.
  • [8. Evaluation Method] can be used as an evaluation method for evaluating the efficacy for acute promyelocytic leukemia as described later in the section.
  • a primer and / or a probe for detecting a polynucleotide according to an embodiment of the present invention and 2) a polypeptide according to an embodiment of the present invention And at least one selected from a probe for detecting
  • [7. Test method] can be used for a test method including a detection step of detecting a polynucleotide and / or a polypeptide in a sample obtained from a subject as described later in the section.
  • primers and probes specifically amplify the polynucleotide to be detected, interact with (specifically bind to) the polypeptide, or interact with (specifically bind to the polypeptide to be detected) And, for example, nucleic acids, antibodies, or peptide probes (not antibodies).
  • the kit according to one embodiment of the present invention may further include at least one of various reagents and devices (buffer solution, test tube, pipette and the like) and instructions for use of the kit, as needed.
  • the instruction manual of the kit may be, for example, the above-mentioned [7. The contents of the method described in the section of [Inspection method] or [8. Evaluation method] are recorded.
  • the test method according to an embodiment of the present invention includes a detection step of detecting the polynucleotide and / or the polypeptide according to an embodiment of the present invention in a sample obtained from a subject.
  • the subject to which the test method according to the present embodiment is applied is a mammal, preferably a human.
  • This test method can be applied to subjects of all ages. It may be preferred that the subject is a patient morphologically diagnosed as being APL. The lower the age of the subject, the better it may be. For example, it is preferable that the subject's age is up to 6 years old or less, and it is more preferable that the subject's age is less than 1 year old or less. By testing at a lower age, it may be possible to provide adequate treatment early.
  • the sample is taken from the subject.
  • the type of sample is not particularly limited as long as it contains at least one of a target protein and a nucleic acid (mRNA as a gene expression product is preferable).
  • the sample includes, for example, a cell sample, a tissue sample, and a body fluid sample. Among them, a body fluid sample is preferable.
  • a body fluid sample As a body fluid sample, a blood sample, a lymph fluid sample, a bone marrow fluid sample and the like can be mentioned, but a blood sample and a bone marrow fluid sample are preferable, and a peripheral blood sample is particularly preferable as a blood sample.
  • the peripheral blood sample can be easily collected, for example, by puncturing the fingertip, and so the burden on the subject is small.
  • the collected sample may be subjected to an operation of extracting a protein or a nucleic acid or an operation of removing an unnecessary component, if necessary, and then subjected to a test.
  • an operation of extracting a protein or a nucleic acid or an operation of removing an unnecessary component, if necessary and then subjected to a test.
  • serum or plasma prepared from the collected blood for the test.
  • the obtained sample may be stored by a method suitable for the type of sample, such as cryopreservation, if necessary.
  • a method suitable for the type of sample such as cryopreservation, if necessary.
  • the sample it is possible to measure the molecular marker to be subjected to the test method at a desired time.
  • the sample as collected may be stored, or a sample (eg, serum or plasma) prepared after collection may be stored.
  • the polynucleotide and / or the polypeptide according to an embodiment of the present invention is detected in the above-mentioned sample.
  • Detecting a polynucleotide and / or a polypeptide is a concept including detecting quantitatively the presence or absence of the polynucleotide and / or the polypeptide.
  • the detection method of the polynucleotide is not particularly limited, but includes a method of amplifying a desired nucleic acid (for example, mRNA as a transcription product) using a nucleic acid amplification technology such as PCR.
  • a nucleic acid amplification technology such as PCR.
  • methods using nucleic acid amplification techniques include quantitative RT-PCR, and methods of directly detecting mRNA include Northern blotting and the like.
  • a method of measuring the expression level of a gene or the like using a nucleic acid chip such as a microarray may be used.
  • classification for classifying objects based on detection results>
  • classification grouping of the objects is performed based on the detection result of the detection step.
  • object classification include the following.
  • Example 1 Grouping into groups A in which a polynucleotide and / or polypeptide according to one embodiment of the present invention is detected in a sample, and group B in which these polynucleotides and / or polypeptides are not detected in a sample Do.
  • the polynucleotide and / or polypeptide according to one embodiment of the present invention is detected in more than a reference value, and in the sample, these polynucleotides and / or polypeptides are within the reference value And the group B.
  • the reference value is, for example, a sample obtained from “healthy person”, “leukemia non-diseased person”, “APL non-affected person”, or “APL patient in which a mutated RARA polypeptide is detected” described later. It is the abundance of the above-mentioned polynucleotide and / or polypeptide.
  • group A samples, whether or not a fusion polypeptide containing a part or the entire length of RARA (retinoic acid receptor- ⁇ ) and / or a polynucleotide encoding the fusion polypeptide is detected or not You may add the item to the inspection item.
  • the fusion polypeptide containing a part or the entire length of RARA is, for example, found in most APL patients such as PML-RARA, PLZF-RARA, NPM-RARA, NUMA1-RARA, or TBL1XR1-RARA.
  • mutant RARA polypeptides, and particularly preferably PML-RARA which occupies a large number of them.
  • Group A is distinguishable from typical APL patients in which a mutated RARA polypeptide is not detected and mutated RARA polypeptide is detected even in APL patients.
  • the test result obtained by performing the above-mentioned test method can be used as one of the diagnostic data when a doctor makes a diagnosis.
  • the doctor can also use this test result to make a diagnosis regarding the determination of the treatment method, the presence or absence of a predisposition to the target disease (APL, etc.), the presence or absence of onset, or the degree of progression.
  • the examination method according to an embodiment of the present invention is applicable to a method of selecting a therapeutic method for APL.
  • APL patients belonging to group A above are all-trans retinoic acid (ATRA) and / or arsenous acid (ATO) compared to typical APL patients (APL patients in which a mutated RARA polypeptide is detected).
  • Administration tends to be less effective.
  • ATRA all-trans retinoic acid
  • ATO arsenous acid
  • administration tends to be less effective.
  • early hematopoietic stem cell transplantation is also a treatment option.
  • An example of a treatment other than the above ATRA and / or ATO administration is a treatment wherein a retinoid other than ATRA is administered.
  • a retinoid other than ATRA is administered.
  • it is a therapeutic method of administering isotretinoin which is one of retinoids other than ATRA.
  • Therapeutics that administer isotretinoin may be particularly effective for APL patients belonging to Group A above.
  • An evaluation method according to an embodiment of the present invention is [2. Polynucleotide according to an embodiment of the present invention] [4. Polypeptide], or [5. Transformant (transformed cell) and Method for Producing Transformant] The method for evaluating the efficacy against APL using the transformed cell described in the section.
  • the target of evaluation is, for example, a candidate for a therapeutic drug for APL, a candidate substance such as a lead compound, or a candidate for a therapy.
  • Those to be evaluated are, for example, candidate substances such as therapeutic drug candidates and lead compounds for APL for which treatment with ATRA and / or ATO is difficult to be successful, or candidates for therapeutic methods.
  • the formation of a homodimer of the above polypeptide is suppressed, the activation of retinoic acid pathway by administration of ATRA is improved, or the above-mentioned cells (especially, blood cells or cells of immune system)
  • Candidate substances and therapeutic candidates that bring about a result such as promotion of differentiation are evaluated as having efficacy.
  • the formation of a homodimer of the above polypeptide is promoted, the activation of retinoic acid pathway by ATRA administration is suppressed, or the above cells (especially, blood cells or cells of immune system)
  • Candidate substances and treatment candidates that bring about a result such as suppression of differentiation are evaluated as ineffective.
  • the formation of homodimers and the like of the polypeptide may also be evaluated using transformed cells. That is, a transformed cell is prepared, and this transformed cell is cultured. Then, candidates for the candidate substance and the therapeutic method may be applied to the transformed cells in culture, and the obtained results may be evaluated.
  • the therapeutic agent for APL not involving RARA according to an embodiment of the present invention contains a retinoid other than ATRA as an active ingredient, and the APL therapeutic agent according to an embodiment of the present invention is effective for isotretinoin Contains as an ingredient.
  • the APL therapeutic agent according to the present invention does not have the risk of inhibiting the activity of the active ingredients, and as long as it is not harmful to the patient being administered It may contain one or more therapeutically active substances having a therapeutic action or other known APL therapeutic active ingredients.
  • the APL therapeutic may also contain a pharmaceutically acceptable carrier that is not harmful to the subject.
  • Carriers which can be used may be in solid, semi-solid or liquid form. For example, water, an electrolyte solution, a sugar solution etc. are mentioned.
  • the APL therapeutic may include an adjuvant.
  • Adjuvants include lubricants, stabilizers, preservatives, emulsifiers, thickeners (thickeners), colorants, flavors (flavoring agents), excipients, moisturizers, disintegrants, surface active agents Preservatives, buffers, flavoring agents, suspending agents, emulsifiers, solubilizers and lubricants, and the like.
  • Dosage forms include tablets, capsules, pills, granules, powders, syrups, emulsions, suppositories, injections, troches and other known forms.
  • the preparation for oral administration is any of tablets, capsules, pills, granules, powders, solutions and syrups, and any of tablets, capsules and granules. Is more preferable, and is more preferably a tablet.
  • it may be formulated as parenteral administration preparations such as injections and suppositories.
  • it may be a freeze-dried preparation.
  • Administration route Routes of administration include intravenous, subcutaneous, intradermal, intraarterial, intraperitoneal, intramuscular, oral cavity, transrectal, and the like. In addition, it may be either oral or parenteral. Intravenous, subcutaneous, intradermal, intraarterial, intraperitoneal, intramuscular, etc. parenteral administration is preferred. From the viewpoint of ease of administration and the like, oral administration may be preferable.
  • the dose (therapeutically effective dose), the number of administrations and the administration period of the APL therapeutic agent may be appropriately set according to the patient's age, sex, weight, symptoms, degree of disease, administration route and the like.
  • the administration subject of the therapeutic agent of the present invention is, like the subject to which the test method of the present invention is applied, a mammal, preferably a human.
  • the subject in one embodiment is a patient morphologically diagnosed as being APL.
  • the administration subject in a more preferred embodiment is a patient diagnosed with APL not involving RARA, more preferably diagnosed with APL without RARA translocation, or diagnosed with APL with RARB translocation I am a patient.
  • the patient is an APL patient classified as belonging to Group A by the above-described examination method according to the present invention.
  • the age of the subject of administration is not particularly limited.
  • the present invention includes any of the following aspects. ⁇ 1> The polynucleotide described in any one of the following 1) to 5): 1) A polynucleotide encoding a fusion polypeptide of a portion or the entire length of RARB (retinoic acid receptor- ⁇ ) and a portion or the entire length of TBL1XR1; 2) A polynucleotide encoding a fusion polypeptide of a part or full length of RARB and a part or full length of TBL1XR1 generated by mutual translocation; 3) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1, which is a polynucleotide encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 1; 4) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1 which encodes
  • ⁇ 2> A polypeptide encoded by any one of the polynucleotides shown in ⁇ 1> above.
  • the inspection method including the detection process which detects the polynucleotide as described in said ⁇ 1>, and / or the polypeptide as described in said ⁇ 2> in the sample acquired from ⁇ 3> object.
  • the inspection method as described in ⁇ 3> which classifies the said object based on the detection result of the ⁇ 4> above-mentioned detection process.
  • the polynucleotide is classified into the group A in which the polynucleotide of ⁇ 1> and / or the polypeptide of ⁇ 2> is detected and the group B in which the polynucleotide is not detected.
  • ⁇ 6> A fusion polypeptide containing a part or the entire length of RARA (retinoic acid receptor- ⁇ ) and / or a polynucleotide encoding the fusion polypeptide is not detected in the sample in the group A, ⁇ 5 The inspection method as described in>.
  • ⁇ 7> The test method according to any one of ⁇ 3> to ⁇ 6>, wherein the sample is blood or bone marrow fluid.
  • a method of selecting a therapeutic method for acute promyelocytic leukemia (APL), which comprises the subjects belonging to the group A classified by the test method according to any one of ⁇ 5> to ⁇ 7> A method of administering all-trans retinoic acid (ATRA) in a larger amount than usual, or selecting a treatment other than ATRA and / or ATO administration.
  • ATRA all-trans retinoic acid
  • ⁇ 10> A cell into which the polynucleotide according to ⁇ 1> is introduced so as to be expressible.
  • the evaluation method which evaluates the effectiveness with respect to acute promyelocytic leukemia using the polynucleotide as described in ⁇ 11> said ⁇ 1>, the polypeptide as described in ⁇ 2>, or the cell as described in ⁇ 9>.
  • ⁇ 12> A kit comprising a primer and / or a probe for detecting the polynucleotide according to ⁇ 1> or a probe for detecting the polypeptide according to ⁇ 2>.
  • ⁇ 13> A therapeutic agent for acute promyelocytic leukemia, in which RARA is not involved, which comprises isotretinoin as an active ingredient.
  • RNA transcripts of TBL1XR1-RARB were confirmed by RT-PCR.
  • Total RNA was extracted from tumor BMNC using RNeasy minikit (Qiagen).
  • cDNA was synthesized using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (TAKARA).
  • a CS-Z2800-02 (3xMYC) expression vector with RARB and an EX-B0004-M07 (3xHA) expression vector with PML-RARA were purchased from GCP.
  • Full-length TBL1XR1-RARB was cloned into pGEM T easy vector (Promega) and CS-Z-2800-02, EX-B0004-M07, PMXs-Puroretrovirus expression vector (CBL) and PMYs-IRES-GFP expression vector It was subcloned into Cell biolabs).
  • RARB was subcloned into EX-B0004-M07 and PMXs-Puroretrovirus expression vectors.
  • PML-RARA was subcloned into CS-Z2800-02, PMXs-Puroretrovirus expression vector and PMYs-IRES-GFP expression vector. Wild-type RARA was cloned from normal PBMC using PrimeSTAR GXL DNA Polymerase (TAKARA) and subcloned into EX-B004-M07.
  • TAKARA PrimeSTAR GXL DNA Polymerase
  • HEK 293 cells and 293 FT cells were maintained in Dulbecco's Modified Eagle Medium (Sigma-Aldrich: D6429) supplemented with 10% fetal bovine serum (FBS) (BioSera: FB-1365).
  • FBS fetal bovine serum
  • U937 cells were maintained in RPMI 1640 Medium (Sigma-Aldrich: R8758) supplemented with 10% FBS. All were incubated at 37 ° C. in a humidified atmosphere of 5% CO 2 . All-trans retinoic acid (ATRA), Am80 and arsenic trioxide (ATO) were purchased from Wako. polybrane was purchased from Sigma-Aldrich. Puromycin was purchased from Gibco.
  • HEK 293 cells were transiently transfected with expression vectors carrying MYC or HA tagged proteins using Fugene HD (promega). After 24 hours, 1 ⁇ M ATRA, Am80 and 0.1 ⁇ M ATO were added and incubated for another 24 hours. The cells are harvested, washed once with D (-) PBS (Wako) and lysed on ice for 15 minutes in 1 ⁇ RIPA buffer (10 mM NaPO 4 , 150 mM NaCl, 1% Triton-X + cOmplete protease inhibitor (Roche diagnostics)) I did.
  • Myc-tagged mouse monoclonal antibody (Myc-mAb) (Cell Signaling) and protein G agarose beads (Cell Signaling) were added to all lysates and incubated at 4 ° C. for 4 hours. These lysates were centrifuged, washed three times with 1 ⁇ RIPA buffer, and proteins were extracted with 3 ⁇ Sample buffer (with 2-mercaptoethanol) (Wako). For immunoblot analysis, extracted proteins were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membrane (GE Healthcare).
  • the proteins were divided into primary antibody (Myc-mAb, HA-Tag rabbit monoclonal antibody (HA-mAb) (Cell Signaling)) and secondary antibody (horseradish peroxidase (HRP) conjugated-anti mouse IgG antibody (DAKO), HRP conjugated- Analyzed by immunoblot using anti-rabbit IgG antibody (DAKO)). Blots were detected using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and visualized using LAS 4000 (GE Healthcare).
  • TBL1XR1-RARB The transcriptional activity of TBL1XR1-RARB was analyzed by luciferase assay.
  • RARE cignal reporter Qiagen
  • a transcription factor responsive construct containing a transcription factor responsive construct and a constitutively expressing Renilla luciferase construct
  • each EX-B0004-M07 expression vector MOCK, RARB, RARA, TBL1XR1-RARB, PML-RARA, TBL1XR1-RARB + RARA , TBL1XR1-RARB + RARB
  • Luciferase assay was performed Dual Luciferase Reporter Assay System (Promega) is used in accordance with the instruction manual, ARVO TM X LIGHT: was detected by (PerkinElmer 2030 Luminescence Reader). Transfection efficiency was normalized by the ratio of firefly luciferase to sea mite luciferase.
  • ATRA-treated or untreated U937 cell lines are incubated with FITC-anti-human CD11b antibody (BioLegend), EC800 flow cytometer (Sony) It evaluated by).
  • FITC-anti-human CD11b antibody BioLegend
  • EC800 flow cytometer Nony
  • FITC isotype-matched mouse IgG1 antibody was used as a negative control.
  • the percentage of CD11b positive cells was analyzed using FlowJo software.
  • cytospin slides 0 and 5 days after ATRA treatment were stained with Wright Giemsa staining solution.
  • Leukocyte cell G binding indicated that it did not have t (15; 17) (q22; q21), and the break-apart fluorescence in situ hybridization (FISH) analysis for RARA was negative ( Figure 1) C and D).
  • FISH fluorescence in situ hybridization
  • TBL1XR1-RARB Influence of TBL1XR1-RARB on retinoic acid pathway
  • the repeated TBL1XR1-RARB fusion had a similar structure to TBL1XR1-RARA and PML-RARA (A in FIG. 2) (Chen Y, Li S, Zhou C, et al., Blood 2014; 124: 936- 45).
  • TBL1XR1-RARB, PML-RARA, RARB and RARA were transduced into 293T cell lines.
  • RARA / RARB is a transcription factor that regulates the retinoic acid pathway activated by retinoic acid.
  • TBL1XR1-RARB The transcriptional activity of TBL1XR1-RARB is smaller compared to RARB, and this fusion has a dominant negative effect on wild-type RARB and suppressed the activation of the retinoic acid pathway in response to ATRA (FIG. 2) B). In addition, the TBL1XR1-RARB fusion had a dominant negative effect on wild-type RARA (FIG. 2C).
  • wild type RARB differentiated the U937 cell line with an increase in expression of CD11b, but TBL1XR1-RARB did not differentiate the U937 cell line (FIG. 2D). Although ATRA differentiated the T937XR1-RARB transduced U937 cell line, the effect was much less than that of PML-RARA.
  • TBL1XR1-RARB Although homodimerization of TBL1XR1-RARB was attenuated by the therapeutic amount of ATRA, the effect was partial. ATRA and tamibarotene (synthetic retinoic acid) had a strong effect on PML-RARA, but the effect of tamibarotene on TBL1XR1-RARB was also insufficient. Of course, ATO has no effect on TBL1XR1-RARB (FIG. 2E). This is because ATO targets PML moieties that interfere with multimerization and induce PML-RARA degradation.
  • retinoids ATRA, Am80, acitretin
  • alitretinoin Alitretinoin
  • bexarotene Bexarotene
  • isotretinoin Isotretinoin
  • tazarotene Tazarotene
  • the cells were further cultured for 7 days to obtain bone marrow cells.
  • the bone marrow cells were administered with the same type of retinoid as that previously administered, and cultured for another 7 days.
  • the percentage of GFP-positive cells and the percentage of cells expressing high CD66 were calculated 24 days after the start of culture.
  • FIG. 4 shows the effect of various retinoid additions on PML-RARA and TBL1XR1-RARB.
  • A shows the percentage of GFP-positive cells on day 24 of culture. The value of each retinoid addition sample when the measured value of the cell which added DMSO as control was set to 1 was compared.
  • B shows the percentage (%) of CD66 high expressing cells on day 24 of culture.
  • isotretinoin in particular, may be effective for the treatment of APL.
  • the present invention can be used, for example, for elucidating an onset mechanism of APL in which RARA is considered not to be involved, and for developing a therapeutic method and a therapeutic agent thereof.

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Abstract

To provide a novel polynucleotide relating to APL and utilization thereof, one embodiment of the present invention pertains to a polynucleotide which encodes a fused polypeptide of a part or the full length of RARB with a part or the full length of TBL1XR1, or a polynucleotide which encodes a RARB mutant caused by translocation.

Description

新規なポリヌクレオチド及びその利用Novel polynucleotide and use thereof
　本発明は、急性前骨髄球性白血病に関連する新規なポリヌクレオチド、及びその利用に関する。 The present invention relates to novel polynucleotides related to acute promyelocytic leukemia, and uses thereof.
　急性前骨髄球性白血病（ＡＰＬ）は、急性骨髄性白血病（ＡＭＬ）の５～１０％を構成する。ＡＭＬは、独特な形態学的特徴によって定義されており、典型的にはアズール性顆粒を含む異常な前骨髄球を伴う。ＡＰＬのゲノム的な根本は、前骨髄球性白血病遺伝子（ＰＭＬ遺伝子）とレチノイン酸レセプターα（ＲＡＲＡ）遺伝子との間の融合を引き起こす相互転座t(15;17)(q22;q21)によって特徴付けられる（非特許文献１～３）。ＰＭＬ－ＲＡＲＡを有するＡＰＬ細胞は、全トランスレチノイン酸（ＡＴＲＡ；オールトランス型レチノイン酸と同義）及び三酸化ヒ素（ＡＴＯ；亜ヒ酸と同義）に対して良好に応答し（非特許文献４～６）、近年の臨床試験では、ＰＭＬ－ＲＡＲＡを伴う典型的なＡＰＬについて優れた結果を達成している。 Acute promyelocytic leukemia (APL) constitutes 5-10% of acute myeloid leukemia (AML). AML is defined by unique morphological features and is typically associated with abnormal promyelocytes, which contain azulotic granules. The genomic root of APL is characterized by a reciprocal translocation t (15; 17) (q22; q21) that causes a fusion between the promyelocytic leukemia gene (PML gene) and the retinoic acid receptor alpha (RARA) gene (Non-Patent Documents 1 to 3). APL cells having PML-RARA respond well to all-trans retinoic acid (ATRA; synonymous with all-trans retinoic acid) and arsenic trioxide (ATO; synonymous with arsenite) (Non-patent document 4) 6) Recent clinical trials have achieved excellent results for typical APL with PML-RARA.
　少数のＡＰＬの事例（５％未満）ではＰＭＬ－ＲＡＲＡを欠いているが、その多くはＲＡＲＡと他のパートナー（ＰＬＺＦ、ＮＰＭ、ＮＵＭＡ１、又はＴＢＬ１ＸＲ１など）との間の相互転座を有する。結局のところ、ＡＰＬに関連する既知の転座の多くが、ＲＡＲＡに関係する。 Although a few APL cases (less than 5%) lack PML-RARA, many have reciprocal translocations between RARA and other partners (such as PLZF, NPM, NUMA1, or TBL1XR1). After all, many of the known translocations associated with APL are associated with RARA.
　しかしながら、集中的なゲノム解析によっても、残りのＡＰＬの事例においてＲＡＲＡの再配列を検出することができず、このＲＡＲＡが関与していないと考えられるＡＰＬの基礎をなす分子メカニズムは未解明である。 However, intensive genomic analysis has also failed to detect RARA rearrangements in the remaining APL cases, and the underlying molecular mechanisms of APL that this RARA may not be involved have not been elucidated. .
　本発明の一態様は、ＡＰＬに関連する新規なポリヌクレオチド、及びその利用を提供することを目的とする。 One aspect of the present invention aims to provide novel polynucleotides related to APL, and uses thereof.
　本発明者らは、ＲＡＲＡ転座を伴わないＡＰＬの事例に焦点を当てて鋭意研究を行った。その結果、ＲＡＲＡ陰性ＡＰＬ患者の大部分においてレチノイン酸レセプターβ（ＲＡＲＢ）の新規な反復融合を見出した。 The inventors conducted intensive studies focusing on the case of APL without RARA translocation. As a result, a novel repeat fusion of retinoic acid receptor beta (RARB) was found in the majority of RARA negative APL patients.
　すなわち、上記の課題を解決するために、本発明は以下の何れかの態様を含んでなる。
＜１＞　以下の１）～５）の何れかに記載されるポリヌクレオチド：
１）ＲＡＲＢ（レチノイン酸受容体‐β）の一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
２）相互転座によって生じた、ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
３）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
４）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
５）転座によって生じた、変異ＲＡＲＢをコードするポリヌクレオチド。
＜２＞　イソトレチノインを有効成分として含む、ＲＡＲＡが関与していない急性前骨髄性白血病の治療薬。 That is, in order to solve the above-mentioned subject, the present invention comprises any one of the following modes.
<1> The polynucleotide described in any one of the following 1) to 5):
1) A polynucleotide encoding a fusion polypeptide of a portion or the entire length of RARB (retinoic acid receptor-β) and a portion or the entire length of TBL1XR1;
2) A polynucleotide encoding a fusion polypeptide of a part or full length of RARB and a part or full length of TBL1XR1 generated by mutual translocation;
3) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1, which is a polynucleotide encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 1;
4) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1 which encodes a polypeptide represented by an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 Polynucleotide;
5) A polynucleotide encoding a mutated RARB produced by translocation.
<2> A therapeutic agent for acute promyelocytic leukemia, in which RARA is not involved, which comprises isotretinoin as an active ingredient.
　本発明の一態様によれば、ＡＰＬに関連する新規なポリヌクレオチド、及びその利用を提供することができる。これにより、例えば、ＲＡＲＡが関与していないと考えられるＡＰＬの発症メカニズムの解明や、その治療法や治療薬の開発等に利用をすることができる。 According to one aspect of the present invention, novel polynucleotides related to APL, and uses thereof can be provided. Thereby, for example, it can be used for elucidation of the onset mechanism of APL in which RARA is considered not to be involved, development of a therapeutic method or a therapeutic agent thereof, and the like.
ＲＡＲＡ転座を伴わないＡＰＬ事例（ＵＰＮ１）の形態学的、細胞遺伝学的及びゲノム的な特徴を示す図である。FIG. 1 shows morphological, cytogenetic and genomic characteristics of APL cases (UPN1) without RARA translocation. in vitroにおける転写活性、分化及びホモ二量体化に対するＴＢＬ１ＸＲ１－ＲＡＲＢの影響を示す図である。FIG. 5 shows the influence of TBL1XR1-RARB on in vitro transcriptional activity, differentiation and homodimerization. ex vivoにおける分化及び増殖に対するＴＢＬ１ＸＲ１－ＲＡＲＢの影響を示す図である。FIG. 5 shows the influence of TBL1XR1-RARB on differentiation and proliferation ex vivo. ＰＭＬ－ＲＡＲＡ及びＴＢＬ１ＸＲ１－ＲＡＲＢに対する各種のレチノイド添加の影響を示す図である。FIG. 5 shows the effects of various retinoid additions on PML-RARA and TBL1XR1-RARB.
　以下、本発明の実施の形態について、詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
　〔１．用語の説明〕
　本明細書において、「Ａ及び／又はＢ」とは、Ａ及びＢと、Ａ又はＢとの両方を意図した表現である。 [1. Explanation of terms]
In the present specification, “A and / or B” are expressions intended for both A and B and A or B.
　本明細書において、「タンパク質」は、「ポリペプチド」とも換言できる。「タンパク質」は、アミノ酸がペプチド結合してなる構造を含むが、さらに、例えば、糖鎖、又はイソプレノイド基などの構造を含んでいてもよい。「タンパク質」は、特に明記しない場合は、天然に存在するアミノ酸と同様に機能することができる、天然に存在するアミノ酸の既知の類似体を含有するポリペプチドを包含する。 In the present specification, "protein" can also be referred to as "polypeptide". The “protein” includes a structure formed by peptide bonding of amino acids, but may further include a structure such as a sugar chain or an isoprenoid group. "Protein," unless otherwise stated, includes polypeptides containing known analogues of naturally occurring amino acids that can function similarly to naturally occurring amino acids.
　本明細書において「核酸」には、任意の単純ヌクレオチド及び／又は修飾ヌクレオチドからなるポリヌクレオチド、例えばｃＤＮＡ、ｍＲＮＡ、全ＲＮＡ、ｈｎＲＮＡ、等が含まれる。「修飾ヌクレオチド」には、イノシン、アセチルシチジン、メチルシチジン、メチルアデノシン、メチルグアノシンを含むリン酸エステルの他、紫外線や化学物質の作用で後天的に発生し得るヌクレオチドも含まれる。 As used herein, "nucleic acid" includes polynucleotides consisting of any simple nucleotides and / or modified nucleotides, such as cDNA, mRNA, total RNA, hnRNA and the like. "Modified nucleotides" include inosine, acetyl cytidine, methyl cytidine, methyl adenosine, and phosphate esters including methyl guanosine, as well as nucleotides that can be generated by the action of ultraviolet light and chemicals.
　本明細書において「遺伝子」は、「ポリヌクレオチド」、「核酸」又は「核酸分子」と交換可能に使用される。「ポリヌクレオチド」はヌクレオチドの重合体を意味する。 As used herein, "gene" is used interchangeably with "polynucleotide", "nucleic acid" or "nucleic acid molecule". "Polynucleotide" means a polymer of nucleotides.
　本明細書での用語「遺伝子」には、２本鎖ＤＮＡのみならず、それを構成するセンス鎖及びアンチセンス鎖といった各１本鎖ＤＮＡやＲＮＡ（ｍＲＮＡ等）を包含する。 The term "gene" as used herein includes not only double-stranded DNA but also single-stranded DNA and RNA (such as mRNA) such as the sense strand and antisense strand that constitute it.
　「ＤＮＡ」には、例えばクローニングや化学合成技術、又はそれらの組み合わせで得られるようなｃＤＮＡやゲノムＤＮＡ等が含まれる。すなわち、ＤＮＡとは、動物等のゲノム中に含まれる形態であるイントロンなどの非コード配列を含む「ゲノム」形ＤＮＡであってもよいし、また逆転写酵素やポリメラーゼを用いてｍＲＮＡを経て得られるｃＤＮＡ、すなわちイントロンなどの非コード配列を含まない「転写」形ＤＮＡであってもよい。 "DNA" includes, for example, cDNA and genomic DNA as obtained by cloning, chemical synthesis techniques, or a combination thereof. That is, the DNA may be a "genomic" form DNA including non-coding sequences such as introns which are contained in the genome of animals etc., or may be obtained through mRNA using reverse transcriptase or polymerase. Or a "transcribed" form of DNA that does not contain non-coding sequences such as introns.
　本明細書において「急性前骨髄球性白血病（ＡＰＬ：Acute Promyelocytic Leukemia）」とは、急性骨髄性白血病の一種に位置づけられ、前骨髄球ががん化する白血病の総称である。急性前骨髄球性白血病は、急性前骨髄性白血病とも称される。 In the present specification, “Acute Promyelocytic Leukemia” (APL) is a kind of acute myeloid leukemia, and is a generic term for leukemia in which promyelocytes become cancerous. Acute promyelocytic leukemia is also referred to as acute promyelocytic leukemia.
　本明細書において「健常者」とは、ＡＰＬを含む一切の疾患に罹患していない状態にあると認められる、正常な個体を指す。本明細書において「白血病非罹患者」とは、少なくとも白血病（ＡＰＬを含む）に罹患していない状態にあると認められる個体を指し、「ＡＰＬ非罹患者」とは、少なくともＡＰＬに罹患していない状態にあると認められる個体を指す。 As used herein, "healthy person" refers to a normal individual who is found to be free of any disease, including APL. As used herein, "leukemia non-affected person" refers to an individual who is at least found to be in a state not suffering from leukemia (including APL), and "APL non-affected person" is at least suffering from APL. Refers to an individual who is found to be absent.
　本明細書において「発症のリスク（危険度）を有する」とは、発症には至っていないが、発症の可能性が健常者と比べて高いことを意図しており、ＡＰＬの発症のリスクを有するとは、ＡＰＬの前段階でありＡＰＬに至る可能性の高い状態であることも包含する。 In the present specification, "having the risk (risk) of onset" is intended to mean that the possibility of onset is higher than that of a healthy person, although it does not lead to onset, and has a risk of onset of APL. That includes the situation before APL and likely to reach APL.
　本明細書において「検査する」又は「検査」とは、医師による同定（診断）を必須としない、検査の対象（「被験者」と称する場合もあり、対象は好ましくはヒトである）における、対象疾患（ＡＰＬ等）の素因の有無、発症の有無、又は進行度等の検査を指す。得られた検査結果は、医師によってなされる診断の一材料になりうる。なお、ＡＰＬの素因の有無を検査するとは、将来的にＡＰＬを発症する可能性を検査することも含む概念である。一方、ＡＰＬの発症の有無を検査するとは、ＡＰＬを発症しているか否かについて検査することを指す。 As used herein, the term "test" or "test" refers to a subject in a subject of a test (sometimes referred to as a "subject", preferably a human being) that does not require identification (diagnosis) by a doctor. Refers to testing for the presence or absence of a predisposition to a disease (such as APL), the presence or absence of onset, or the degree of progression. The obtained test result can be one of the materials for diagnosis made by a doctor. In addition, examining the presence or absence of the predisposing factor of APL is a concept including examining the possibility of developing APL in the future. On the other hand, examining the presence or absence of the onset of APL refers to examining whether or not the onset of APL has occurred.
　本明細書において「治療」とは、対象疾患の症状を完治又は軽減させること、対象疾患の症状の悪化を抑制すること、対象疾患の発症を抑制すること、又は遅延させることを含む。すなわち、被験者が対象疾患を発症していない場合の「予防」を含む。 As used herein, “treatment” includes completely curing or reducing the symptoms of the target disease, suppressing the aggravation of the symptoms of the target disease, suppressing or delaying the onset of the target disease. That is, it includes "prevention" when the subject does not develop the target disease.
　〔２．本発明の一実施形態に係るポリヌクレオチド〕
　このポリヌクレオチドは、具体的には、以下の（１）～（５）の何れかに記載のポリヌクレオチドである。 [2. Polynucleotide according to one embodiment of the present invention]
Specifically, this polynucleotide is a polynucleotide described in any of the following (1) to (5).
　(１）ＲＡＲＢ（レチノイン酸受容体‐β）の一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド。 (1) A polynucleotide encoding a fusion polypeptide of a portion or the entire length of RARB (retinoic acid receptor-β) and a portion or the entire length of TBL1XR1.
　(２）相互転座によって生じた、ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド。なお、後述する実施例で得られたポリペプチドの一つも、相互転座によって生じた新規なポリペプチドである。 (2) A polynucleotide encoding a fusion polypeptide of a part or full length of RARB and a part or full length of TBL1XR1 generated by mutual translocation. In addition, one of the polypeptides obtained in the Examples described later is also a novel polypeptide generated by mutual translocation.
　(３）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド。なお、配列番号１のアミノ酸配列で示されるポリペプチドとは、後述する実施例で得られたポリペプチドの一つである。 (3) A polynucleotide encoding a fusion polypeptide of a portion or full length of RARB and a portion or full length of TBL1XR1, which polypeptide is represented by the amino acid sequence of SEQ ID NO: 1. The polypeptide represented by the amino acid sequence of SEQ ID NO: 1 is one of the polypeptides obtained in the examples described later.
　(４）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；この配列同一性は８５％以上であることが好ましく、８８％以上であることがより好ましく、９０％以上であることがさらに好ましく、９５％以上であることが特に好ましく、９６％以上、９７％以上、９８％以上、或いは９９％以上であることが特に好ましい。
なお、配列番号１のアミノ酸配列において１～１００個のアミノ酸が置換、欠失、挿入、及び／又は付加されたアミノ酸配列を有し、配列番号１のアミノ酸配列を有するポリペプチドと同等の機能を有するポリペプチドをコードするポリヌクレオチドであってもよい。ここで、置換、欠失、挿入、及び／又は付加されたアミノ酸の個数は、１～５４個であることが好ましく、１～２７個であることがより好ましく、１～２０個であることがさらに好ましく、１～１５個、１～１０個或いは１～６個であることが特に好ましい。或いは、上記（３）に記載のポリヌクレオチドと相補的な配列からなるポリヌクレオチドに対して、ストリンジェントな条件下においてハイブリダイズし、配列番号１のアミノ酸配列を有するポリペプチドと同等の機能を有するポリペプチドをコードするポリヌクレオチドであってもよい。このポリヌクレオチドは、上記（３）に記載のポリヌクレオチドの塩基配列に対して８５％以上の配列同一性を有することが好ましい。当該配列同一性は、８８％以上であることが好ましく、９０％以上であることがより好ましく、９５％以上であることがさらに好ましく、９６％以上、９７％以上、９８％以上、或いは９９％以上であることが特に好ましい。
なお、上記のストリンジェントな条件下とは、例えば、参考文献[Molecular cloning-a Laboratory manual 2nd edition(Sambrookら、1989)]に記載の条件などが挙げられる。ストリンジェントな条件下とは、より具体的には例えば、６×ＳＳＣ（１×ＳＳＣの組成：０．１５Ｍ塩化ナトリウム、０．０１５Ｍクエン酸ナトリウム、ｐＨ７．０）、０．５％ＳＤＳ、５×デンハート及び１００ｍｇ／ｍＬニシン精子ＤＮＡを含む溶液にプローブとともに６５℃で８～１６時間インキュベートし、ハイブリダイズさせる条件が挙げられる。 (4) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1, which is represented by an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 This sequence identity is preferably 85% or more, more preferably 88% or more, still more preferably 90% or more, particularly preferably 95% or more, 96% It is particularly preferable that the content is 97% or more, 98% or more, or 99% or more.
The amino acid sequence of SEQ ID NO: 1 has an amino acid sequence in which 1 to 100 amino acids have been substituted, deleted, inserted and / or added, and functions the same as the polypeptide having the amino acid sequence of SEQ ID NO: 1 It may be a polynucleotide encoding a polypeptide which it has. Here, the number of amino acids substituted, deleted, inserted and / or added is preferably 1 to 54, more preferably 1 to 27, and 1 to 20. More preferably, 1 to 15, 1 to 10 or 1 to 6 is particularly preferable. Alternatively, it hybridizes under stringent conditions to a polynucleotide consisting of a sequence complementary to the polynucleotide described in (3) above, and has the same function as a polypeptide having the amino acid sequence of SEQ ID NO: 1 It may be a polynucleotide encoding a polypeptide. This polynucleotide preferably has a sequence identity of 85% or more to the nucleotide sequence of the polynucleotide described in (3) above. The sequence identity is preferably 88% or more, more preferably 90% or more, still more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. It is particularly preferable to be the above.
The above-mentioned stringent conditions include, for example, the conditions described in the reference document [Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)]. More specifically, under stringent conditions, for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 Conditions for hybridization by incubating a solution containing × Denhardt and 100 mg / mL herring sperm DNA at 65 ° C. for 8 to 16 hours with a probe may be mentioned.
　なお、上記（１）～（４）に記載のＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドには、例えば、ＲＡＲＢのModulating regionの一部（Ｎ末端側の部分）が、ＬｉｓＨドメインを含むＴＢＬ１ＸＲ１のＮ末端側の断片で置き換えられたものや、ＲＡＲＢのModulating regionの一部（Ｎ末端側の部分）が、ＬｉｓＨドメイン及びＦ－ｂｏｘ－like domainを含むＴＢＬ１ＸＲ１のＮ末端側の断片で置き換えられたもの、等が挙げられる。 The fusion polypeptide of a part or the entire length of RARB and a part or the entire length of TBL1XR1 described in (1) to (4) above includes, for example, a part (the N-terminal side) of the modulating region of RARB. Is replaced with a TBL1XR1 N-terminal fragment containing a LisH domain, or a part (N-terminal part) of the modulating region of RARB is a TBL1XR1 N containing a LisH domain and an F-box-like domain. Those which have been replaced by fragments on the terminal side, and the like can be mentioned.
　(５）転座によって生じた、変異ＲＡＲＢをコードするポリヌクレオチド。なお、後述する実施例で得られたポリペプチドの一つも、一つの遺伝子内での転座、或いは二つの遺伝子間での相互転座によって生じた新規なポリペプチドである。 (5) A polynucleotide encoding a mutant RARB, which is generated by translocation. In addition, one of the polypeptides obtained in the examples described later is also a novel polypeptide produced by translocation within one gene or mutual translocation between two genes.
　本発明の一実施形態に係るポリヌクレオチドを取得する（単離する）方法は、特に限定されるものではないが、例えば、上記ポリヌクレオチドの塩基配列の一部と特異的にハイブリダイズするプローブを調製し、ゲノムＤＮＡライブラリー又はｃＤＮＡライブラリーをスクリーニングすればよい。或いは、本発明に係るポリヌクレオチドを、ホスホロアミダイト法などの核酸合成法に従って合成してもよい。 The method for obtaining (isolating) the polynucleotide according to one embodiment of the present invention is not particularly limited, and, for example, a probe that specifically hybridizes with a part of the base sequence of the above-mentioned polynucleotide It may be prepared and screened for genomic DNA library or cDNA library. Alternatively, the polynucleotide according to the present invention may be synthesized according to a nucleic acid synthesis method such as the phosphoroamidite method.
　また、本発明の一実施形態に係るポリヌクレオチドを取得する方法として、ＰＣＲなどの核酸増幅法を用いる方法を挙げることができる。例えば、当該ポリヌクレオチドのｃＤＮＡのうち、５’側及び３’側の配列（又はその相補配列）の中からそれぞれプライマーを調製し、これらプライマーを用いてゲノムＤＮＡ又はｃＤＮＡなどを鋳型にしてＰＣＲなどを行い、両プライマー間に挟まれるＤＮＡ領域を増幅する。これによって、本発明の一実施形態に係るポリヌクレオチドを含むＤＮＡ断片を大量に取得できる。 Further, as a method for obtaining the polynucleotide according to an embodiment of the present invention, a method using a nucleic acid amplification method such as PCR can be mentioned. For example, a primer is prepared from each of the 5 'and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and these primers are used for PCR using genomic DNA or cDNA as a template. And amplify the DNA region sandwiched between the two primers. Thereby, a large amount of DNA fragments containing the polynucleotide according to one embodiment of the present invention can be obtained.
　〔３．組み換えベクター〕
　本発明の一実施形態に係るポリヌクレオチド（例えばＤＮＡ）は、適当なベクター中に挿入して、組み換えベクターとして利用してもよい。ベクターの種類は、プラスミドのような自律的に複製するベクターでもよいし、或いは、宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、宿主細胞の染色体と共に複製されるものであってもよい。 [3. Recombinant vector]
The polynucleotide (for example, DNA) according to one embodiment of the present invention may be inserted into an appropriate vector and used as a recombinant vector. The type of vector may be an autonomously replicating vector such as a plasmid, or it may be integrated into the host cell's genome when introduced into the host cell and replicated together with the host cell's chromosomes. Good.
　上記ベクターは、好ましくは発現ベクターである。発現ベクターにおいて、本発明の一実施形態に係るポリヌクレオチドは、例えば、プロモータ配列などの、転写に必要な要素が、機能的に連結されている。プロモータ配列は宿主細胞において転写活性を示すＤＮＡ配列である。用いるプロモータ配列の種類は、宿主細胞の種類に応じて適宜選択すればよい。 The vector is preferably an expression vector. In an expression vector, a polynucleotide according to an embodiment of the present invention is functionally linked to elements necessary for transcription, such as, for example, a promoter sequence. A promoter sequence is a DNA sequence that shows transcriptional activity in a host cell. The type of promoter sequence to be used may be appropriately selected according to the type of host cell.
　発現ベクターにおいて、本発明の一実施形態に係るポリヌクレオチドは、必要に応じて、適切なターミネータに機能的に結合されてもよい。適切なターミネータの種類は、宿主細胞の種類に応じて適宜選択すればよい。 In the expression vector, the polynucleotide according to one embodiment of the present invention may be functionally linked to an appropriate terminator, as needed. An appropriate type of terminator may be appropriately selected depending on the type of host cell.
　本発明の一実施形態に係る組み換えベクターは、さらに、ポリアデニレーションシグナル、転写エンハンサ配列、又は翻訳エンハンサ配列などの要素を有していてもよい。 The recombinant vector according to one embodiment of the present invention may further have an element such as a polyadenylation signal, a transcription enhancer sequence, or a translation enhancer sequence.
　本発明の一実施形態に係る組み換えベクターは、さらに、該ベクターの宿主細胞内での複製を可能にするＤＮＡ配列を有していてもよい。 The recombinant vector according to one embodiment of the present invention may further have a DNA sequence that allows the vector to replicate in a host cell.
　本発明の一実施形態に係る組み換えベクターはさらに選択マーカーを有していてもよい。選択マーカーとしては、例えば、アンピシリン、カナマイシン、テトラサイクリン、クロラムフェニコール、ネオマイシン若しくはヒグロマイシンのような薬剤に対する、薬剤耐性遺伝子を挙げることができる。 The recombinant vector according to one embodiment of the present invention may further have a selectable marker. A selectable marker can include, for example, a drug resistant gene to a drug such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin or hygromycin.
　〔４．ポリペプチド〕
　本発明の一実施形態に係るポリペプチドは、上述した本発明の一実施形態に係るポリヌクレオチドの何れかによってコードされているポリペプチドである。なお、配列番号１のアミノ酸配列を有するポリペプチドは実施例にて得られたポリペプチドである。当該ポリペプチドは、例えば、ホモ二量体を形成し、ＡＴＲＡ投与によるレチノイン酸経路の活性化を抑制し、上記細胞（特に、前骨髄球を含む、血液系の細胞や免疫系の細胞）の分化を抑制するという機能を有すると推定される。 [4. Polypeptide]
The polypeptide according to an embodiment of the present invention is a polypeptide encoded by any of the polynucleotides according to an embodiment of the present invention described above. The polypeptide having the amino acid sequence of SEQ ID NO: 1 is the polypeptide obtained in the example. The polypeptide forms, for example, a homodimer and suppresses the activation of the retinoic acid pathway by ATRA administration, and the above-mentioned cells (especially, blood cells and cells of immune system including promyelocytes). It is presumed to have the function of suppressing differentiation.
　上記ポリペプチドの由来は限定されず、天然供給源より単離されても、化学合成されても、或いは、遺伝子組み換え技術を用いて産生されてもよい。より具体的には、当該ポリペプチドは、単離精製された天然のポリペプチド、化学合成されたポリペプチド、及び、遺伝子組み換え技術に基づいて宿主細胞から産生されたポリペプチドをその範疇に含む。なお、宿主細胞については「形質転換体」を説明する欄で詳述する。 The origin of the polypeptide is not limited, and may be isolated from natural sources, chemically synthesized, or produced using genetic engineering techniques. More specifically, the polypeptide includes, in its category, an isolated and purified natural polypeptide, a chemically synthesized polypeptide, and a polypeptide produced from a host cell based on genetic engineering technology. The host cell will be described in detail in the section describing “transformant”.
　なお、上述した、本発明の一実施形態に係るポリヌクレオチド及びポリペプチドは、ＡＰＬの原因遺伝子及びタンパク質の一つと推定されるため、例えば、ＡＰＬの発症メカニズムの解明、ＡＰＬの治療法（治療薬を含む）の開発、ＡＰＬの診断、並びに、ＡＰＬの治療法の選択等に利用可能である。 In addition, since the polynucleotide and polypeptide according to the embodiment of the present invention described above are presumed to be one of the causative gene and protein of APL, for example, elucidation of the onset mechanism of APL, therapy for APL (therapeutic agent Development of APL, diagnosis of APL, and selection of therapy for APL.
　〔５．形質転換体（形質転換細胞）、及び形質転換体の作製方法〕
　本発明の一実施形態に係るポリヌクレオチド又は組み換えベクターを適当な宿主細胞に、発現可能に導入することによって形質転換体を作製することができる。 [5. Transformant (transformed cell) and method for producing transformant]
A transformant can be produced by introduceably expressing a polynucleotide or a recombinant vector according to one embodiment of the present invention into a suitable host cell.
　なお、以下の説明において、本発明の一実施形態に係るポリヌクレオチド及び組み換えベクターを、「外来（foreign）核酸分子」と総称する。外来核酸分子を宿主細胞に導入する方法は、下記に例示をする通り、宿主細胞の種類に応じて選択すればよい。また、本発明の一実施形態に係る形質転換体の子孫を得る方法も、形質転換体の種類に応じて選択すればよい。 In the following description, the polynucleotide and the recombinant vector according to one embodiment of the present invention are generically referred to as "foreign nucleic acid molecule". The method for introducing the foreign nucleic acid molecule into the host cell may be selected according to the type of host cell, as exemplified below. Also, the method of obtaining the offspring of the transformant according to one embodiment of the present invention may be selected according to the type of transformant.
　宿主細胞としては、例えば、細菌細胞、酵母細胞、酵母細胞以外の真菌細胞、及び高等真核細胞などが挙げられる。高等真核細胞としては、例えば、植物細胞、動物細胞が挙げられる。動物細胞としては、昆虫細胞、両生類細胞、爬虫類細胞、鳥類細胞、魚類細胞、哺乳動物細胞などが挙げられる。 Host cells include, for example, bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Higher eukaryotic cells include, for example, plant cells and animal cells. Animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, mammalian cells and the like.
　細菌細胞の例としては、バチルス又はストレプトマイセスなどのグラム陽性菌；大腸菌などのグラム陰性菌；が挙げられる。これら細菌細胞の形質転換は、例えば、プロトプラスト法、又はコンピテント細胞を用いる方法などにより行えばよい。 Examples of bacterial cells include gram positive bacteria such as Bacillus or Streptomyces; Gram negative bacteria such as E. coli. Transformation of these bacterial cells may be performed by, for example, a protoplast method or a method using competent cells.
　酵母細胞の例としては、サッカロマイセス又はシゾサッカロマイセスに属する細胞が挙げられる。外来核酸分子の、酵母宿主への導入方法としては、例えば、エレクトロポレーション法、スフェロブラスト法、酢酸リチウム法などを挙げることができる。 Examples of yeast cells include cells belonging to Saccharomyces or Schizosaccharomyces. Examples of methods for introducing foreign nucleic acid molecules into a yeast host include electroporation, spheroplast method, lithium acetate method and the like.
　酵母細胞以外の真菌細胞の例としては、糸状菌の細胞が挙げられる。宿主細胞として糸状菌を用いる場合、外来核酸分子を宿主染色体に組み込んで組み換え宿主細胞を得ることによって、形質転換を行うことができる。外来核酸分子の宿主染色体への組み込みは、例えば、相同組換え又は異種組換えにより行うことができる。 Examples of fungal cells other than yeast cells include filamentous fungal cells. When filamentous fungi are used as host cells, transformation can be carried out by incorporating foreign nucleic acid molecules into the host chromosome to obtain recombinant host cells. Integration of foreign nucleic acid molecules into the host chromosome can be performed, for example, by homologous recombination or heterologous recombination.
　昆虫細胞の例としては、例えば、カイコの細胞などが挙げられる。宿主細胞として昆虫細胞を用いる場合、外来核酸分子及びバキュロウイルスを昆虫細胞に共導入して昆虫細胞の培養上清中に組換えバキュロウイルスを得る。次いで、組換えバキュロウイルスを昆虫細胞に感染させる。共導入の方法としては、例えば、リン酸カルシウム法又はリポフェクション法などを挙げることができる。 Examples of insect cells include, for example, silkworm cells. When insect cells are used as host cells, foreign nucleic acid molecules and baculovirus are co-introduced into insect cells to obtain recombinant baculovirus in the culture supernatant of insect cells. The insect cells are then infected with the recombinant baculovirus. As a method of co-introduction, for example, calcium phosphate method or lipofection method can be mentioned.
　哺乳動物細胞の例としては、ＨＥＫ２９３細胞、ＨｅＬａ細胞、ＣＯＳ細胞、ＢＨＫ細胞、ＣＨＬ細胞、ＣＨＯ細胞などが挙げられる。また、ＡＰＬが前骨髄球をがん化させる疾患であることを考慮すれば、用いる細胞は、血液系の細胞であるか、免疫系の細胞であることが好ましい場合がある。血液系の細胞や、免疫系の細胞としては、例えば、Ｊｅｋｏ－1細胞、Ｒａｊｉ細胞、Ｒａｍｏｓ細胞等のＢ細胞株；Ｊｕｒｋａｔ細胞、ＭＯＬＴ－４細胞、ＴＧ４０細胞、Ｕ－９３７細胞等のＴ細胞株；ＫＵ８１２‐Ｆ細胞、ＲＢＬ－２Ｈ３細胞等の好塩基球細胞株；ＨＭＣ－１細胞、Ｋｕ８１２細胞、ＲＢＬ細胞等の顆粒球細胞株；ＭＣＬ－５細胞、ＭＯＬＴ－４細胞等のリンパ芽球細胞株；ＮＢ－４細胞、ＴＨＰ－１細胞等の好中球様細胞株；４１６Ｂ細胞、ＭＯＬＭ－１４細胞、ＴＨＰ－１細胞等のマクロファージ細胞株；ＢＪＡＢ細胞、ＣＭＬ細胞、Ｄａｕｄｉ細胞等の白血球細胞株；Ａ２０細胞、ＢＣ－３細胞、ＣＡ４６細胞、ＨＣＴ１１６細胞、ＳＴ－４８６細胞等のリンパ腫細胞株；ＥＳＢ細胞、ＴＣＬＢ細胞等のリンパ芽球腫細胞株；ＨＬ－６０細胞、Ｋ５６２細胞等の白血病細胞株；等が挙げられる。本発明において用いる血液系の細胞は、分化したものであっても未分化なものであってもよい。未分化な細胞の例としては、例えば、造血前駆細胞、造血幹細胞などが挙げられる。さらに、未分化な造血細胞としては、人工多能性幹細胞（iPS細胞）、胚性幹細胞（ES細胞）、血液細胞（臍帯血細胞など）、分化細胞をリプログラムした細胞等を例示することができる。
哺乳動物細胞の形質転換には、例えば、エレクトロポレーション法、リン酸カルシウム法、リポフェクション法などを用いることができる。 Examples of mammalian cells include HEK 293 cells, HeLa cells, COS cells, BHK cells, CHL cells, CHO cells and the like. Also, in view of the fact that APL is a disease that causes promyelocytes to become cancerous, it may be preferable that the cells used be blood cells or cells of the immune system. Blood cells and cells of the immune system, for example, B cell lines such as Jeko-1 cells, Raji cells, Ramos cells etc .; T cells such as Jurkat cells, MOLT-4 cells, TG40 cells, U-937 cells etc. Strains; basophil cell lines such as KU812-F cells, RBL-2H3 cells; granulocyte cell lines such as HMC-1 cells, Ku812 cells, RBL cells; lymphoblasts such as MCL-5 cells, MOLT-4 cells Cell lines; Neutrophil-like cell lines such as NB-4 cells, THP-1 cells; Macrophage cell lines such as 416 B cells, MOLM-14 cells, THP-1 cells; Leukocytes such as BJAB cells, CML cells, Daudi cells Cell line; Lymphoma cell line such as A20 cell, BC-3 cell, CA46 cell, HCT116 cell, ST-486 cell, etc .; Lymphoblast such as ESB cell, TCLB cell Tumor cell lines; leukemia cell lines such as HL-60 cells and K562 cells; The blood cells used in the present invention may be differentiated or undifferentiated. Examples of undifferentiated cells include, for example, hematopoietic progenitor cells, hematopoietic stem cells and the like. Furthermore, as undifferentiated hematopoietic cells, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), blood cells (such as cord blood cells), cells obtained by reprogramming differentiated cells, and the like can be exemplified. .
For transformation of mammalian cells, for example, electroporation, calcium phosphate method, lipofection method and the like can be used.
　植物細胞の形質転換には、例えば、エレクトロポレーション法、アグロバクテリウム法、パーティクルガン法などを用いることができる。 For transformation of plant cells, for example, an electroporation method, an Agrobacterium method, a particle gun method or the like can be used.
　上記の形質転換体は、導入された外来核酸分子の発現が可能な条件下で、培養又は育成する。次いで、必要に応じて、形質転換体から、本発明の一実施形態に係るポリペプチドを単離精製する。或いは、必要に応じて、形質転換体そのものを、各種の評価等に用いる。 The transformant described above is cultured or grown under conditions that allow expression of the introduced foreign nucleic acid molecule. Then, if necessary, the polypeptide according to one embodiment of the present invention is isolated and purified from the transformant. Alternatively, if necessary, the transformant itself is used for various evaluations and the like.
　なお、形質転換体は、細胞に限定されない。すなわち、形質転換体は、例えば、外来核酸分子で形質転換された組織、器官、及び個体であってもよい。ただし、細胞以外の形質転換体は、非ヒト由来のものであることが好ましい場合があり、特に個体は非ヒト由来のものであることが好ましい。 The transformant is not limited to cells. That is, transformants may be, for example, tissues, organs, and individuals transformed with foreign nucleic acid molecules. However, it may be preferable that the non-cell transformant is of non-human origin, and in particular, the individual is preferably of non-human origin.
　〔６．キット〕
　本発明の一実施形態に係るキットの一例では、１）本発明の一実施形態に係るポリヌクレオチド、２）本発明の一実施形態に係るポリペプチド、及び、３）本発明の一実施形態に係る細胞、から選択される少なくとも一つを含む。このキットは、例えば、〔８．評価方法〕の欄で後述するような、急性前骨髄性白血病に対する有効性を評価する評価方法に用いることが出来る。 [6. kit〕
In an example of a kit according to an embodiment of the present invention, 1) a polynucleotide according to an embodiment of the present invention, 2) a polypeptide according to an embodiment of the present invention, and 3) an embodiment of the present invention And at least one selected from such cells. For example, [8. Evaluation Method] can be used as an evaluation method for evaluating the efficacy for acute promyelocytic leukemia as described later in the section.
　本発明の一実施形態に係るキットの他の例では、１）本発明の一実施形態に係るポリヌクレオチドを検出するプライマー及び／又はプローブ、並びに、２）本発明の一実施形態に係るポリペプチドを検出するプローブ、から選択される少なくとも一つを含む。このキットは、例えば、〔７．検査方法〕の欄で後述するような、対象より取得した試料におけるポリヌクレオチド、及び／又は、ポリペプチドを検出する検出工程を含む検査方法に用いることが出来る。上述のプライマー及びプローブは、検出対象であるポリヌクレオチドを特異的に増幅する、当該ポリペプチドと相互作用（特異的に結合）する、又は、検出対象であるポリペプチドと相互作用（特異的に結合）するものが挙げられ、例えば、核酸、抗体、又はペプチドプローブ（抗体ではない）である。 In another example of a kit according to an embodiment of the present invention, 1) a primer and / or a probe for detecting a polynucleotide according to an embodiment of the present invention, and 2) a polypeptide according to an embodiment of the present invention And at least one selected from a probe for detecting For example, [7. Test method] can be used for a test method including a detection step of detecting a polynucleotide and / or a polypeptide in a sample obtained from a subject as described later in the section. The above-mentioned primers and probes specifically amplify the polynucleotide to be detected, interact with (specifically bind to) the polypeptide, or interact with (specifically bind to the polypeptide to be detected) And, for example, nucleic acids, antibodies, or peptide probes (not antibodies).
　本発明の一実施形態に係るキットは、さらに必要に応じて、各種試薬及び器具（緩衝溶液、試験管及びピペットなど）ならびにキットの使用説明書などの少なくとも１つを備えていてもよい。なお、キットの使用説明書には、例えば、上述の〔７．検査方法〕又は〔８.評価方法〕の欄で説明した、方法の内容が記録されている。 The kit according to one embodiment of the present invention may further include at least one of various reagents and devices (buffer solution, test tube, pipette and the like) and instructions for use of the kit, as needed. The instruction manual of the kit may be, for example, the above-mentioned [7. The contents of the method described in the section of [Inspection method] or [8. Evaluation method] are recorded.
　〔７．検査方法〕
　本発明の一実施形態に係る検査方法は、対象より取得した試料における、本発明の一実施形態に記載のポリヌクレオチド及び／又はポリペプチドを検出する検出工程を含む。 [7. Inspection method〕
The test method according to an embodiment of the present invention includes a detection step of detecting the polynucleotide and / or the polypeptide according to an embodiment of the present invention in a sample obtained from a subject.
　＜対象＞
　本実施形態に係る検査方法が適用される対象は、哺乳動物であり、中でもヒトであることが好ましい。この検査方法は、あらゆる年齢の対象に適用することができる。対象は、ＡＰＬであると形態学的に診断された患者であることが好ましい場合がある。なお、被験者の年齢は低いほど好ましい場合があり、例えば満６歳以下の幼児期までであることが好ましく、満１歳以下の乳児期までであることがより好ましい場合がある。より低い年齢で検査することにより、早期に適切な治療法を提供することが可能となり得る。 <Target>
The subject to which the test method according to the present embodiment is applied is a mammal, preferably a human. This test method can be applied to subjects of all ages. It may be preferred that the subject is a patient morphologically diagnosed as being APL. The lower the age of the subject, the better it may be. For example, it is preferable that the subject's age is up to 6 years old or less, and it is more preferable that the subject's age is less than 1 year old or less. By testing at a lower age, it may be possible to provide adequate treatment early.
　＜試料＞
　試料は、対象から採取される。試料の種類としては、特に限定されず、対象のタンパク質及び核酸（遺伝子発現産物としてのｍＲＮＡが好ましい）の少なくとも一方を含んでいればよい。試料としては、例えば、細胞試料、組織試料、体液試料が挙げられ、中でも体液試料が好ましい。体液試料としては、血液試料、リンパ液試料、骨髄液試料等が挙げられるが、血液試料及び骨髄液試料が好ましく、血液試料としては特に末梢血試料が好ましい。末梢血試料は、例えば指先への穿刺等によって容易に採取が可能であるから対象の負担が少ない。 <Sample>
The sample is taken from the subject. The type of sample is not particularly limited as long as it contains at least one of a target protein and a nucleic acid (mRNA as a gene expression product is preferable). The sample includes, for example, a cell sample, a tissue sample, and a body fluid sample. Among them, a body fluid sample is preferable. As a body fluid sample, a blood sample, a lymph fluid sample, a bone marrow fluid sample and the like can be mentioned, but a blood sample and a bone marrow fluid sample are preferable, and a peripheral blood sample is particularly preferable as a blood sample. The peripheral blood sample can be easily collected, for example, by puncturing the fingertip, and so the burden on the subject is small.
　採取された試料は、必要に応じてタンパク質又は核酸を抽出する操作を行ったり、不要な成分を除去する操作を行ってから、検査に供してもよい。例えば、血液試料を用いる場合は、採取した血液から調製した血清又は血漿を検査に用いることが好ましい。 The collected sample may be subjected to an operation of extracting a protein or a nucleic acid or an operation of removing an unnecessary component, if necessary, and then subjected to a test. For example, when using a blood sample, it is preferable to use serum or plasma prepared from the collected blood for the test.
　また、得られた試料は、必要に応じて凍結保存等の、試料の種類に適した方法で保存してもよい。試料は、保存することにより所望の時期に検査方法の対象となる分子マーカーを測定することができる。なお、保存の際には、採取したままの状態の試料を保存してもよく、また採取した後に調製した試料（例えば血清又は血漿）を保存してもよい。 In addition, the obtained sample may be stored by a method suitable for the type of sample, such as cryopreservation, if necessary. By storing the sample, it is possible to measure the molecular marker to be subjected to the test method at a desired time. During storage, the sample as collected may be stored, or a sample (eg, serum or plasma) prepared after collection may be stored.
　＜検出工程＞
　検出工程では、上記試料における、本発明の一実施形態に係るポリヌクレオチド及び／又はポリペプチドを検出する。ポリヌクレオチド及び／又はポリペプチドを検出するとは、これらの有無を検出する事の他、定量的に検出することも含む概念である。 <Detection process>
In the detection step, the polynucleotide and / or the polypeptide according to an embodiment of the present invention is detected in the above-mentioned sample. Detecting a polynucleotide and / or a polypeptide is a concept including detecting quantitatively the presence or absence of the polynucleotide and / or the polypeptide.
　ポリヌクレオチドの検出方法としては、特に限定されないが、ＰＣＲ法等の核酸増幅技術を用いて所望の核酸（例えば、転写産物たるｍＲＮＡ）を増幅する手法を含む方法が挙げられる。核酸増幅技術を用いた方法としては、例えば、定量ＲＴ‐ＰＣＲが挙げられ、直接ｍＲＮＡを検出する方法としてはノーザンブロット法等が挙げられる。あるいは、マイクロアレイ等の核酸チップを用いた、遺伝子の発現量の測定方法等であってもよい。 The detection method of the polynucleotide is not particularly limited, but includes a method of amplifying a desired nucleic acid (for example, mRNA as a transcription product) using a nucleic acid amplification technology such as PCR. Examples of methods using nucleic acid amplification techniques include quantitative RT-PCR, and methods of directly detecting mRNA include Northern blotting and the like. Alternatively, a method of measuring the expression level of a gene or the like using a nucleic acid chip such as a microarray may be used.
　ポリペプチドの検出方法としては、当該ポリペプチドに対して特異的な抗体を用いた方法（例えば、各種のＥＬＩＳＡ（Enzyme-Linked Immuno Sorbent Assay、酵素結合免疫吸着アッセイ）法）、液体カラムクロマトグラフィー及び質量分析法などが挙げられる。 As a method of detecting a polypeptide, a method using an antibody specific to the polypeptide (for example, various ELISA (Enzyme-Linked Immuno Sorbent Assay) method, liquid column chromatography, and the like) Mass spectrometry etc. are mentioned.
　＜検出の結果に基づき対象を分類する分類工程＞
　分類工程では、上記検出工程の検出結果に基づいて、上記対象の分類（グループ分け）を行う。対象の分類としては、以下の例が挙げられる。 <Classification process for classifying objects based on detection results>
In the classification step, classification (grouping) of the objects is performed based on the detection result of the detection step. Examples of object classification include the following.
　（例１）　試料において、本発明の一実施形態に係るポリヌクレオチド及び／又はポリペプチドが検出される群Ａと、試料において、これらポリヌクレオチド及び／又はポリペプチドが検出されない群Ｂとにグループ分けする。 (Example 1) Grouping into groups A in which a polynucleotide and / or polypeptide according to one embodiment of the present invention is detected in a sample, and group B in which these polynucleotides and / or polypeptides are not detected in a sample Do.
　（例２）　試料において、本発明の一実施形態に係るポリヌクレオチド及び／又はポリペプチドが基準値よりも多く検出される群Ａと、試料において、これらポリヌクレオチド及び／又はポリペプチドが基準値内である群Ｂとにグループ分けする。なお、基準値は、例えは、「健常者」、「白血病非罹患者」、「ＡＰＬ非罹患者」、或いは、後述する「変異ＲＡＲＡポリペプチドが検出されるＡＰＬ患者」から取得した試料における、上記ポリヌクレオチド及び／又はポリペプチドの存在量である。 (Example 2) In a sample, the polynucleotide and / or polypeptide according to one embodiment of the present invention is detected in more than a reference value, and in the sample, these polynucleotides and / or polypeptides are within the reference value And the group B. The reference value is, for example, a sample obtained from “healthy person”, “leukemia non-diseased person”, “APL non-affected person”, or “APL patient in which a mutated RARA polypeptide is detected” described later. It is the abundance of the above-mentioned polynucleotide and / or polypeptide.
　なお、さらに、上記群Ａの試料において、ＲＡＲＡ（レチノイン酸受容体‐α）の一部又は全長を含む融合ポリペプチド、及び／又は、当該融合ポリペプチドをコードするポリヌクレオチドが検出されるか否かを検査項目に加えてもよい。なお、ＲＡＲＡの一部又は全長を含む融合ポリペプチドとは、例えば、ＰＭＬ－ＲＡＲＡ、ＰＬＺＦ－ＲＡＲＡ、ＮＰＭ－ＲＡＲＡ、ＮＵＭＡ１－ＲＡＲＡ、又は、ＴＢＬ１ＸＲ１－ＲＡＲＡ等の、大部分のＡＰＬ患者に認められる、変異ＲＡＲＡポリペプチドが挙げられ、特に多数を占めるＰＭＬ－ＲＡＲＡであることが好ましい。一例において、群Ａでは、ＡＰＬ患者であっても変異ＲＡＲＡポリペプチドが検出されず、変異ＲＡＲＡポリペプチドが検出される典型的なＡＰＬ患者と区別可能である。 Furthermore, in the above-mentioned group A samples, whether or not a fusion polypeptide containing a part or the entire length of RARA (retinoic acid receptor-α) and / or a polynucleotide encoding the fusion polypeptide is detected or not You may add the item to the inspection item. The fusion polypeptide containing a part or the entire length of RARA is, for example, found in most APL patients such as PML-RARA, PLZF-RARA, NPM-RARA, NUMA1-RARA, or TBL1XR1-RARA. And mutant RARA polypeptides, and particularly preferably PML-RARA, which occupies a large number of them. In one example, Group A is distinguishable from typical APL patients in which a mutated RARA polypeptide is not detected and mutated RARA polypeptide is detected even in APL patients.
　＜診断への応用＞
　上記の検査方法を行うことによって得られた検査結果は、医師による診断を行う際の診断資料の１つとして利用することができる。医師はこの検査結果も利用して、治療方法の決定、対象疾患（ＡＰＬ等）の素因の有無、発症の有無、又は進行度等に関する診断を行うこともできる。 <Application to diagnosis>
The test result obtained by performing the above-mentioned test method can be used as one of the diagnostic data when a doctor makes a diagnosis. The doctor can also use this test result to make a diagnosis regarding the determination of the treatment method, the presence or absence of a predisposition to the target disease (APL, etc.), the presence or absence of onset, or the degree of progression.
　＜治療法の選択への応用＞
　本発明の一実施形態に係る検査方法は、ＡＰＬに対する治療法を選択する方法に応用可能である。例えば、上記群Ａに属するＡＰＬ患者は、典型的なＡＰＬ患者（変異ＲＡＲＡポリペプチドが検出されるＡＰＬ患者）と比較して、オールトランス型レチノイン酸（ＡＴＲＡ）及び／又は亜ヒ酸（ＡＴＯ)の投与が奏功しにくい傾向がある。従って、例えば、上記検査方法によって分類される群Ａに属する対象に対して、通常よりも多量のＡＴＲＡを投与するか、ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法を選択することが出来る。群Ａに属する対象に対しては、早期の造血幹細胞移植も治療法の選択の一つである。一方、上記検査方法によって分類される群Ｂに属するＡＰＬ患者に対して、通常と同等量のＡＴＲＡ及び／又はＡＴＯを投与する治療法を選択することもできる。 <Application to selection of therapy>
The examination method according to an embodiment of the present invention is applicable to a method of selecting a therapeutic method for APL. For example, APL patients belonging to group A above are all-trans retinoic acid (ATRA) and / or arsenous acid (ATO) compared to typical APL patients (APL patients in which a mutated RARA polypeptide is detected). Administration tends to be less effective. Thus, for example, for subjects belonging to group A classified by the above-mentioned test method, it is possible to administer a larger amount of ATRA than usual, or to select a therapeutic method other than ATRA and / or ATO administration. For subjects belonging to group A, early hematopoietic stem cell transplantation is also a treatment option. On the other hand, it is also possible to select a treatment method of administering an equivalent amount of ATRA and / or ATO to the APL patients belonging to the group B classified by the above-mentioned test method.
　上記ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法の一例はＡＴＲＡ以外のレチノイドを投与する治療法である。例えば、ＡＴＲＡ以外のレチノイドの一種であるイソトレチノインを投与する治療法である。イソトレチノインを投与する治療法は特に上記群Ａに属するＡＰＬ患者に有効であり得る。 An example of a treatment other than the above ATRA and / or ATO administration is a treatment wherein a retinoid other than ATRA is administered. For example, it is a therapeutic method of administering isotretinoin which is one of retinoids other than ATRA. Therapeutics that administer isotretinoin may be particularly effective for APL patients belonging to Group A above.
　〔８.評価方法〕
　本発明の一実施形態に係る評価方法は、〔２．本発明の一実施形態に係るポリヌクレオチド〕欄に記載のポリヌクレオチド、〔４．ポリペプチド〕欄に記載のポリペプチド、又は、〔５．形質転換体（形質転換細胞）、及び形質転換体の作製方法〕欄に記載の形質転換細胞を用いて、ＡＰＬに対する有効性を評価する方法である。 [8. Evaluation method]
An evaluation method according to an embodiment of the present invention is [2. Polynucleotide according to an embodiment of the present invention] [4. Polypeptide], or [5. Transformant (transformed cell) and Method for Producing Transformant] The method for evaluating the efficacy against APL using the transformed cell described in the section.
　評価の対象となるものは、例えば、ＡＰＬの治療薬候補やリード化合物等の候補物質、或いは、治療法の候補である。評価の対象となるものは、例えば、ＡＴＲＡ及び／又はＡＴＯによる治療が奏功しにくいＡＰＬに対する治療薬候補やリード化合物等の候補物質、或いは、治療法の候補である。 The target of evaluation is, for example, a candidate for a therapeutic drug for APL, a candidate substance such as a lead compound, or a candidate for a therapy. Those to be evaluated are, for example, candidate substances such as therapeutic drug candidates and lead compounds for APL for which treatment with ATRA and / or ATO is difficult to be successful, or candidates for therapeutic methods.
　例えば、上記ポリペプチドのホモ二量体の形成が抑制される、ＡＴＲＡ投与によるレチノイン酸経路の活性化が向上する、或いは、上記細胞（特に血液系の細胞であるか、免疫系の細胞）の分化が促進される等の結果をもたらす候補物質や治療法の候補は、有効性ありと評価される。一方、上記ポリペプチドのホモ二量体の形成が促進される、ＡＴＲＡ投与によるレチノイン酸経路の活性化が抑制される、或いは、上記細胞（特に血液系の細胞であるか、免疫系の細胞）の分化が抑制される等の結果をもたらす候補物質や治療法の候補は、有効性なしと評価される。 For example, the formation of a homodimer of the above polypeptide is suppressed, the activation of retinoic acid pathway by administration of ATRA is improved, or the above-mentioned cells (especially, blood cells or cells of immune system) Candidate substances and therapeutic candidates that bring about a result such as promotion of differentiation are evaluated as having efficacy. On the other hand, the formation of a homodimer of the above polypeptide is promoted, the activation of retinoic acid pathway by ATRA administration is suppressed, or the above cells (especially, blood cells or cells of immune system) Candidate substances and treatment candidates that bring about a result such as suppression of differentiation are evaluated as ineffective.
　なお、ポリペプチドのホモ二量体の形成等についても、形質転換細胞を用いて評価すればよい。すなわち、形質転換細胞を調製し、この形質転換細胞を培養する。そして、培養中の形質転換細胞に対して、候補物質や治療法の候補を適用し、得られた結果を評価すればよい。 The formation of homodimers and the like of the polypeptide may also be evaluated using transformed cells. That is, a transformed cell is prepared, and this transformed cell is cultured. Then, candidates for the candidate substance and the therapeutic method may be applied to the transformed cells in culture, and the obtained results may be evaluated.
　〔１０．ＡＰＬ治療薬〕
　本発明の一実施形態に係る、ＲＡＲＡが関与していないＡＰＬの治療薬は、ＡＴＲＡ以外のレチノイドを有効成分として含んでおり、本発明のある実施形態に係るＡＰＬ治療薬は、イソトレチノインを有効成分として含む。 [10. APL therapeutic agent]
The therapeutic agent for APL not involving RARA according to an embodiment of the present invention contains a retinoid other than ATRA as an active ingredient, and the APL therapeutic agent according to an embodiment of the present invention is effective for isotretinoin Contains as an ingredient.
　（任意の成分）
　本発明に係るＡＰＬ治療薬は、上記の有効成分の他に、有効成分の活性を阻害する虞がなく、投与対象である患者にとって有害でないものである限り、他の疾病、疾患及び状態に対して治療作用を有する治療活性物質又は他の公知のＡＰＬ治療有効成分を１つ以上含んでいてもよい。 (Any ingredient)
In addition to the above-mentioned active ingredients, the APL therapeutic agent according to the present invention does not have the risk of inhibiting the activity of the active ingredients, and as long as it is not harmful to the patient being administered It may contain one or more therapeutically active substances having a therapeutic action or other known APL therapeutic active ingredients.
　また、投与対象に有害でない、薬学的に受容可能な担体を含んでいてもよい。用いることが可能な担体は、固体、半固体、又は液体型の何れであってもよい。例えば、水、電解質液及び糖液等が挙げられる。さらに、ＡＰＬ治療薬は、補助剤を含んでいてもよい。補助剤としては、潤滑剤、安定化剤、防腐剤、乳化剤、増粘剤（粘稠剤）、着色剤、香料（着香剤）、賦形剤、付湿剤、崩壊剤、表面活性剤、保存剤、緩衝剤、矯味剤、懸濁化剤、乳化剤、溶解補助剤及び滑沢剤などが挙げられる。 It may also contain a pharmaceutically acceptable carrier that is not harmful to the subject. Carriers which can be used may be in solid, semi-solid or liquid form. For example, water, an electrolyte solution, a sugar solution etc. are mentioned. In addition, the APL therapeutic may include an adjuvant. Adjuvants include lubricants, stabilizers, preservatives, emulsifiers, thickeners (thickeners), colorants, flavors (flavoring agents), excipients, moisturizers, disintegrants, surface active agents Preservatives, buffers, flavoring agents, suspending agents, emulsifiers, solubilizers and lubricants, and the like.
　（剤型）
　剤型としては、錠剤、カプセル剤、丸剤、顆粒剤、散剤、シロップ剤、乳剤、坐剤、注射剤、トローチ剤及び公知のその他の形態が挙げられる。これらの中でも、例えば、経口投与製剤として、錠剤、カプセル剤、丸剤、顆粒剤、散剤、液剤及びシロップ剤のうちのいずれかであることが好ましく、錠剤、カプセル剤及び顆粒剤のうちのいずれかであることがより好ましく、錠剤であることがさらに好ましい。なお、例えば、注射剤及び坐剤のような非経口投与製剤として製剤されてもよい。さらに凍結乾燥製剤としてもよい。 (Formulation type)
Dosage forms include tablets, capsules, pills, granules, powders, syrups, emulsions, suppositories, injections, troches and other known forms. Among these, for example, it is preferable that the preparation for oral administration is any of tablets, capsules, pills, granules, powders, solutions and syrups, and any of tablets, capsules and granules. Is more preferable, and is more preferably a tablet. In addition, for example, it may be formulated as parenteral administration preparations such as injections and suppositories. Furthermore, it may be a freeze-dried preparation.
　（投与経路）
　投与経路は、静脈内、皮下、皮内、動脈内、腹腔内、筋肉内、経口腔、経直腸経由等が挙げられる。また、経口及び非経口のいずれであってもよい。静脈内、皮下、皮内、動脈内、腹腔内、筋肉内等の非経口投与が好ましい。投与の容易さ等の観点からは、経口投与されることが好ましい場合がある。 (Administration route)
Routes of administration include intravenous, subcutaneous, intradermal, intraarterial, intraperitoneal, intramuscular, oral cavity, transrectal, and the like. In addition, it may be either oral or parenteral. Intravenous, subcutaneous, intradermal, intraarterial, intraperitoneal, intramuscular, etc. parenteral administration is preferred. From the viewpoint of ease of administration and the like, oral administration may be preferable.
　（投与量、投与回数及び投与期間）
　上記ＡＰＬ治療薬の投与量（治療有効量）、投与回数及び投与期間は、患者の年齢、性別、体重、症状、疾患の程度、投与経路等に応じて適宜設定すればよい。 (Dose, frequency and period of administration)
The dose (therapeutically effective dose), the number of administrations and the administration period of the APL therapeutic agent may be appropriately set according to the patient's age, sex, weight, symptoms, degree of disease, administration route and the like.
　（投与対象）
　本発明の治療薬の投与対象は、本発明の検査方法が適用される対象と同様に、哺乳動物であり、中でもヒトであることが好ましい。一実施形態における対象は、ＡＰＬであると形態学的に診断された患者である。より好ましい実施形態における投与対象は、ＲＡＲＡが関与していないＡＰＬと診断された患者であり、さらに好ましくはＲＡＲＡ転座を伴わないＡＰＬと診断された、またはＲＡＲＢ転座を伴うＡＰＬと診断された患者である。ある実施形態では上述の本発明に係る検査方法によって上記群Ａに属すると分類されたＡＰＬ患者である。また、投与対象の年齢は特に限定されない。 (Subject of administration)
The administration subject of the therapeutic agent of the present invention is, like the subject to which the test method of the present invention is applied, a mammal, preferably a human. The subject in one embodiment is a patient morphologically diagnosed as being APL. The administration subject in a more preferred embodiment is a patient diagnosed with APL not involving RARA, more preferably diagnosed with APL without RARA translocation, or diagnosed with APL with RARB translocation I am a patient. In one embodiment, the patient is an APL patient classified as belonging to Group A by the above-described examination method according to the present invention. Also, the age of the subject of administration is not particularly limited.
　〔１１．本発明に係る具体的な態様の例示〕
　本発明は以下の何れかの態様を包含する。
＜１＞　以下の１）～５）の何れかに記載されるポリヌクレオチド：
１）ＲＡＲＢ（レチノイン酸受容体‐β）の一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
２）相互転座によって生じた、ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
３）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
４）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
５）転座によって生じた、変異ＲＡＲＢをコードするポリヌクレオチド。
＜２＞　上記＜１＞に示す何れかのポリヌクレオチドによってコードされているポリペプチド。
＜３＞　対象より取得した試料における、上記＜１＞に記載のポリヌクレオチド、及び／又は、上記＜２＞に記載のポリペプチドを検出する検出工程を含む、検査方法。
＜４＞　上記検出工程の検出結果に基づいて、上記対象の分類を行う、＜３＞に記載の検査方法。
＜５＞　上記対象の分類として、上記試料において、＜１＞に記載のポリヌクレオチド、及び／又は、＜２＞に記載のポリペプチドが検出される群Ａと、検出されない群Ｂとに分類する、＜４＞に記載の検査方法。
＜６＞　ＲＡＲＡ（レチノイン酸受容体‐α）の一部又は全長を含む融合ポリペプチド、及び／又は、当該融合ポリペプチドをコードするポリヌクレオチドが、上記群Ａでは上記試料において検出されない、＜５＞に記載の検査方法。
＜７＞　上記試料は血液又は骨髄液である、＜３＞～＜６＞の何れかに記載の検査方法。＜８＞　急性前骨髄性白血病（ＡＰＬ）に対する治療法を選択する方法であって、＜５＞～＜７＞の何れかに記載の検査方法によって分類される上記群Ａに属する対象に対して、通常よりも多量の、オールトランス型レチノイン酸（ＡＴＲＡ）を投与するか、ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法を選択する、方法。
＜８＞　上記ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法はイソトレチノインを投与する治療法である、上記＜８＞に記載の方法。
＜１０＞　上記＜１＞に記載のポリヌクレオチドが発現可能に導入されている細胞。
＜１１＞　上記＜１＞に記載のポリヌクレオチド、＜２＞に記載のポリペプチド、又は、＜９＞に記載の細胞を用いて、急性前骨髄性白血病に対する有効性を評価する、評価方法。
＜１２＞　上記＜１＞に記載のポリヌクレオチドを検出するプライマー及び／又はプローブを含むか、＜２＞に記載のポリペプチドを検出するプローブを含んでいる、キット。
＜１３＞　イソトレチノインを有効成分として含む、ＲＡＲＡが関与していない急性前骨髄性白血病の治療薬。 [11. Examples of specific embodiments according to the present invention]
The present invention includes any of the following aspects.
<1> The polynucleotide described in any one of the following 1) to 5):
1) A polynucleotide encoding a fusion polypeptide of a portion or the entire length of RARB (retinoic acid receptor-β) and a portion or the entire length of TBL1XR1;
2) A polynucleotide encoding a fusion polypeptide of a part or full length of RARB and a part or full length of TBL1XR1 generated by mutual translocation;
3) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1, which is a polynucleotide encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 1;
4) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1 which encodes a polypeptide represented by an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 Polynucleotide;
5) A polynucleotide encoding a mutated RARB produced by translocation.
<2> A polypeptide encoded by any one of the polynucleotides shown in <1> above.
The inspection method including the detection process which detects the polynucleotide as described in said <1>, and / or the polypeptide as described in said <2> in the sample acquired from <3> object.
The inspection method as described in <3> which classifies the said object based on the detection result of the <4> above-mentioned detection process.
<5> As the classification of the above object, the polynucleotide is classified into the group A in which the polynucleotide of <1> and / or the polypeptide of <2> is detected and the group B in which the polynucleotide is not detected. , The inspection method as described in <4>.
<6> A fusion polypeptide containing a part or the entire length of RARA (retinoic acid receptor-α) and / or a polynucleotide encoding the fusion polypeptide is not detected in the sample in the group A, <5 The inspection method as described in>.
<7> The test method according to any one of <3> to <6>, wherein the sample is blood or bone marrow fluid. <8> A method of selecting a therapeutic method for acute promyelocytic leukemia (APL), which comprises the subjects belonging to the group A classified by the test method according to any one of <5> to <7> A method of administering all-trans retinoic acid (ATRA) in a larger amount than usual, or selecting a treatment other than ATRA and / or ATO administration.
<8> The method according to <8>, wherein the therapeutic method other than the ATRA and / or ATO administration is a therapeutic method of administering isotretinoin.
<10> A cell into which the polynucleotide according to <1> is introduced so as to be expressible.
The evaluation method which evaluates the effectiveness with respect to acute promyelocytic leukemia using the polynucleotide as described in <11> said <1>, the polypeptide as described in <2>, or the cell as described in <9>.
<12> A kit comprising a primer and / or a probe for detecting the polynucleotide according to <1> or a probe for detecting the polypeptide according to <2>.
<13> A therapeutic agent for acute promyelocytic leukemia, in which RARA is not involved, which comprises isotretinoin as an active ingredient.
　以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples will be shown below, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it is needless to say that various aspects are possible as to details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included. It is included in the technical scope of the invention. Also, all of the documents described in the present specification are incorporated by reference.
　〔方法〕
　（ＩＲＢの承認）
　本研究は、国立成育医療研究センターの倫理審査委員会によって承認されている（＃１０３５）。患者の親又は保護者からインフォームドコンセントを得ている。 〔Method〕
(Approved by IRB)
This study has been endorsed by the Ethics Review Board of the National Center for Growth and Medical Research (# 1035). Informed consent is obtained from the patient's parent or guardian.
　（ゲノム解析）
　ＴＢＬ１ＸＲ１－ＲＡＲＢのメッセンジャーＲＮＡ（ｍＲＮＡ）転写物は、ＲＴ－ＰＣＲによって確認した。全ＲＮＡは、RNeasy minikit（Qiagen）を用いて腫瘍ＢＭＮＣから抽出した。ｃＤＮＡは、Superscript III First-Strand Synthesis System for RT-PCR（Invitrogen）を用いて合成した。ＰＣＲ増幅は、PrimeSTAR GXL DNA Polymerase（TAKARA）を用いて行った。 (Genome analysis)
The messenger RNA (mRNA) transcripts of TBL1XR1-RARB were confirmed by RT-PCR. Total RNA was extracted from tumor BMNC using RNeasy minikit (Qiagen). cDNA was synthesized using Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen). PCR amplification was performed using PrimeSTAR GXL DNA Polymerase (TAKARA).
　（コンストラクト）
　ＲＡＲＢを有するCS-Z2800-02 (3xMYC)発現ベクター及びＰＭＬ－ＲＡＲＡを有するEX-B0004-M07 (3xHA)発現ベクターは、ＧＣＰから購入した。全長ＴＢＬ１ＸＲ１－ＲＡＲＢは、pGEM T easy ベクター（Promega）にクローニングし、CS-Z-2800-02、EX-B0004-M07、PMXs-ピューロレトロウイルス発現ベクター（CBL）及びPMYs-IRES-GFP発現ベクター（Cell biolabs）にサブクローニングした。ＲＡＲＢは、EX-B0004-M07及びPMXs-ピューロレトロウイルス発現ベクターにサブクローニングした。ＰＭＬ－ＲＡＲＡは、CS-Z2800-02、PMXs-ピューロレトロウイルス発現ベクター及びPMYs-IRES-GFP発現ベクターにサブクローニングした。野生型ＲＡＲＡは、PrimeSTAR GXL DNA Polymerase（TAKARA）を用いて正常なＰＢＭＣからクローニングし、EX-B004-M07にサブクローニングした。 (construct)
A CS-Z2800-02 (3xMYC) expression vector with RARB and an EX-B0004-M07 (3xHA) expression vector with PML-RARA were purchased from GCP. Full-length TBL1XR1-RARB was cloned into pGEM T easy vector (Promega) and CS-Z-2800-02, EX-B0004-M07, PMXs-Puroretrovirus expression vector (CBL) and PMYs-IRES-GFP expression vector It was subcloned into Cell biolabs). RARB was subcloned into EX-B0004-M07 and PMXs-Puroretrovirus expression vectors. PML-RARA was subcloned into CS-Z2800-02, PMXs-Puroretrovirus expression vector and PMYs-IRES-GFP expression vector. Wild-type RARA was cloned from normal PBMC using PrimeSTAR GXL DNA Polymerase (TAKARA) and subcloned into EX-B004-M07.
　（細胞株及び試薬）
　ＨＥＫ２９３細胞及び２９３ＦＴ細胞は、１０％ウシ胎児血清（ＦＢＳ）（BioSera: FB-1365）を添加したDulbecco’s Modified Eagle Medium（Sigma-Aldrich: D6429）中で維持した。Ｕ９３７細胞は、１０％ＦＢＳを添加したRPMI 1640 Medium（Sigma-Aldrich: R8758）中で維持した。全て５％ＣＯ２の加湿雰囲気中３７℃でインキュベートした。全トランスレチノイン酸（ＡＴＲＡ）、Ａｍ８０及び三酸化ヒ素（ＡＴＯ）は、Wakoから購入した。polybraneはSigma-Aldrichから購入した。ピューロマイシンは、Gibcoから購入した。 (Cell lines and reagents)
HEK 293 cells and 293 FT cells were maintained in Dulbecco's Modified Eagle Medium (Sigma-Aldrich: D6429) supplemented with 10% fetal bovine serum (FBS) (BioSera: FB-1365). U937 cells were maintained in RPMI 1640 Medium (Sigma-Aldrich: R8758) supplemented with 10% FBS. All were incubated at 37 ° C. in a humidified atmosphere of 5% CO 2 . All-trans retinoic acid (ATRA), Am80 and arsenic trioxide (ATO) were purchased from Wako. polybrane was purchased from Sigma-Aldrich. Puromycin was purchased from Gibco.
　（共免疫沈殿及び免疫ブロット分析）
　共免疫沈殿については、ＭＹＣ又はＨＡタグ付きタンパク質を有する発現ベクターをFugene HD（promega）を用いてＨＥＫ２９３細胞に一過的にトランスフェクトした。２４時間後、１μＭ ＡＴＲＡ、Ａｍ８０及び０．１μＭ ＡＴＯを添加し、さらに２４時間インキュベートした。細胞を回収し、D(-)PBS（Wako）で１回洗浄し、氷上で1xRIPA buffer（10mM NaPO4、150mM NaCl、1%Triton-X+cOmplete protease inhibitor（Roche diagnostics））中で１５分間溶解させた。遠心分離後、上清を回収した。次いで、Ｍｙｃタグ付きマウスモノクローナル抗体（Myc-mAb）（Cell Signaling）及びプロテインＧアガロースビーズ（Cell Signaling）を全溶解物に添加し、４℃で４時間インキュベートした。これらの溶解物を遠心分離し、1xRIPA bufferで３回洗浄し、タンパク質を3xSample buffer（2-メルカプトエタノール入り）（Wako）で抽出した。免疫ブロット分析については、抽出したタンパク質を１０％ドデシル硫酸ナトリウムポリアクリルアミドゲル電気泳動ゲル中で分離し、ニトロセルロース膜（GE Healthcare）に転写した。タンパク質を、一次抗体（Myc-mAb、HA-Tagウサギモノクローナル抗体（HA-mAb）（Cell Signaling））及び二次抗体（セイヨウワサビペルオキシダーゼ（ＨＲＰ）共役-抗マウスＩｇＧ抗体（DAKO）、ＨＲＰ共役-抗ウサギＩｇＧ抗体（DAKO））を用いた免疫ブロットによって分析した。ブロットは、Amersham ECL Western Blotting Detection Reagent（GE Healthcare）を用いて検出し、LAS 4000（GE Healthcare）を用いて可視化した。 (Co-immunoprecipitation and immunoblot analysis)
For co-immunoprecipitation, HEK 293 cells were transiently transfected with expression vectors carrying MYC or HA tagged proteins using Fugene HD (promega). After 24 hours, 1 μM ATRA, Am80 and 0.1 μM ATO were added and incubated for another 24 hours. The cells are harvested, washed once with D (-) PBS (Wako) and lysed on ice for 15 minutes in 1 × RIPA buffer (10 mM NaPO 4 , 150 mM NaCl, 1% Triton-X + cOmplete protease inhibitor (Roche diagnostics)) I did. After centrifugation, the supernatant was collected. Then, Myc-tagged mouse monoclonal antibody (Myc-mAb) (Cell Signaling) and protein G agarose beads (Cell Signaling) were added to all lysates and incubated at 4 ° C. for 4 hours. These lysates were centrifuged, washed three times with 1 × RIPA buffer, and proteins were extracted with 3 × Sample buffer (with 2-mercaptoethanol) (Wako). For immunoblot analysis, extracted proteins were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to nitrocellulose membrane (GE Healthcare). The proteins were divided into primary antibody (Myc-mAb, HA-Tag rabbit monoclonal antibody (HA-mAb) (Cell Signaling)) and secondary antibody (horseradish peroxidase (HRP) conjugated-anti mouse IgG antibody (DAKO), HRP conjugated- Analyzed by immunoblot using anti-rabbit IgG antibody (DAKO)). Blots were detected using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) and visualized using LAS 4000 (GE Healthcare).
　（ルシフェラーゼアッセイ）
　ＴＢＬ１ＸＲ１－ＲＡＲＢの転写活性は、ルシフェラーゼアッセイによって分析した。転写因子応答性コンストラクト及び恒常発現ウミシイタケルシフェラーゼコンストラクトを含むRARE cignal reporter（Qiagen）と、各EX-B0004-M07発現ベクター（MOCK、RARB、RARA、TBL1XR1-RARB、PML-RARA、TBL1XR1-RARB+RARA、TBL1XR1-RARB+RARB）を、ＨＥＫ２９３細胞に一過的に共トランスフェクトした。２４時間後に種々の濃度のＡＴＲＡを添加し、さらに２４時間インキュベートした。ルシフェラーゼアッセイは、Dual Luciferase Reporter Assay System（Promega）を取扱説明書に従って用いて行い、ARVOTMX LIGHT（PerkinElmer: 2030 Luminescence Reader）によって検出した。ホタルルシフェラーゼとウミシイタケルルシフェラーゼとの比でトランスフェクション効率を正規化した。 (Luciferase assay)
The transcriptional activity of TBL1XR1-RARB was analyzed by luciferase assay. RARE cignal reporter (Qiagen) containing a transcription factor responsive construct and a constitutively expressing Renilla luciferase construct, and each EX-B0004-M07 expression vector (MOCK, RARB, RARA, TBL1XR1-RARB, PML-RARA, TBL1XR1-RARB + RARA , TBL1XR1-RARB + RARB) were transiently cotransfected into HEK293 cells. After 24 hours, various concentrations of ATRA were added and incubated for another 24 hours. Luciferase assay was performed Dual Luciferase Reporter Assay System (Promega) is used in accordance with the instruction manual, ARVO TM X LIGHT: was detected by (PerkinElmer 2030 Luminescence Reader). Transfection efficiency was normalized by the ratio of firefly luciferase to sea mite luciferase.
　（レトロウイルス産生及び形質導入）
　FugeneHD reagentを用いて、２９３ＦＴ細胞に、各pMXs-ピューロレトロウイルス発現ベクター、gag-pol及びpVSVGコンストラクトをトランスフェクトした。４８時間後、上清を回収し、４℃で１６時間遠心分離することによって濃縮した。polybrane（１０μｇ／ｍＬ）を用いて、Ｕ９３７細胞を用意したレトロウイルス（MOCK、TBL1XR1-RARB、PML-RARA）に感染させ、４８時間後に２μｇ／ｍＬピューロマイシンで選択した。 (Retrovirus production and transduction)
293FT cells were transfected with each pMXs-Puroretrovirus expression vector, gag-pol and pVSVG constructs using Fugene HD reagent. After 48 hours, the supernatant was collected and concentrated by centrifugation at 4 ° C. for 16 hours. U937 cells were infected with prepared retrovirus (MOCK, TBL1XR1-RARB, PML-RARA) using polybrane (10 μg / mL), and selected with 2 μg / mL puromycin 48 hours later.
　（ＦＣＭ及び形態学的分析）
　ＡＴＲＡ処理したＵ９３７細胞株又は未処理のＵ９３７細胞株を、ＦＩＴＣ-抗ヒトＣＤ１１ｂ抗体（BioLegend）と一緒にインキュベートし、EC800 flow cytometer（Sony
）で評価した。ネガティブコントロールとしてFITC isotype-matchedマウスIgG1抗体を用いた。ＣＤ１１ｂ陽性細胞のパーセンテージは、FlowJoソフトウェアを用いて解析した。形態学的分析については、ＡＴＲＡ処理から０日及び５日後のサイトスピンスライドをライトギムザ染色溶液で染色した。 (FCM and morphological analysis)
ATRA-treated or untreated U937 cell lines are incubated with FITC-anti-human CD11b antibody (BioLegend), EC800 flow cytometer (Sony)
It evaluated by). As a negative control, FITC isotype-matched mouse IgG1 antibody was used. The percentage of CD11b positive cells was analyzed using FlowJo software. For morphological analysis, cytospin slides 0 and 5 days after ATRA treatment were stained with Wright Giemsa staining solution.
　〔事例〕
　白血球増加症及び播種性血管内凝固症候群を発症している２才の男児（患者識別名：ＵＰＮ１）。骨髄吸引の結果、アズール性顆粒を伴う細胞芽の存在が明らかとなり、急性前骨髄球性白血病（ＡＰＬ）であると形態学的に診断された（図１のＡ及びＢ）。全トランスレチノイン酸（ＡＴＲＡ）を用いた治療は効果がなく、ＰＭＬ（前骨髄球性白血病）－ＲＡＲＡ（レチノイン酸レセプターα）についてのＰＣＲは陰性であった。白血球細胞のＧバインディングによってt(15;17)(q22;q21)を有さないことが示され、ＲＡＲＡについてのbreak-apart蛍光in situハイブリダイゼーション（ＦＩＳＨ）分析は陰性であった（図１のＣ及びＤ）。多剤併用細胞毒性化学療法後に完全な寛解に達したが、治療完了後に再発し、２回目の寛解において臍帯血移植を行った。移植から２６ヶ月経過した現時点では、疾患の徴候なく生存している。 [Example]
A 2-year-old boy who developed leukocytosis and disseminated intravascular coagulation syndrome (patient identification name: UPN1). As a result of bone marrow aspiration, the presence of cellular buds with azure granules was revealed, and morphologically diagnosed as acute promyelocytic leukemia (APL) (FIG. 1A and B). Treatment with all-trans retinoic acid (ATRA) was ineffective and PCR for PML (promyelocytic leukemia) -RARA (retinoic acid receptor alpha) was negative. Leukocyte cell G binding indicated that it did not have t (15; 17) (q22; q21), and the break-apart fluorescence in situ hybridization (FISH) analysis for RARA was negative (Figure 1) C and D). A complete remission was reached after multidrug cytotoxic chemotherapy, but relapsed after treatment completion, and cord blood transplantation was performed in the second remission. At 26 months after transplantation, the patient is alive without signs of disease.
　さらに、ＦＩＳＨでＲＡＲＡ転座が検出されなかった、ＡＰＬであると形態学的に診断されたさらなる４例を集めた。上述の例と同様に、ＲＡＲＡ転座のない当該４例は、ＡＴＲＡに対して応答せず、また、ＡＭＬとして多剤併用化学療法を施したが、５例中４例では、再発したか、又は寛解に達しなかった。これらの事例の特徴及び臨床経過の詳細は、表１及び補足図１に示されている。事例ＵＰＮ５の臨床経過は、既にIgarashi K, Hori T, Yamamoto M, et al., J Pediatr Hematol Oncol 2015;37:e234-7で報告されている。 In addition, 4 additional cases were collected that were morphologically diagnosed as APL, with no RARA translocation detected in FISH. As in the previous example, the four cases without RARA translocation did not respond to ATRA and also received combination chemotherapy as AML, but in 4 out of 5 cases, did they relapse, Or did not reach remission. The characteristics of these cases and the details of the clinical course are shown in Table 1 and Supplementary Figure 1. The clinical course of the case UPN5 has already been reported in Igarashi K, Hori T, Yamamoto M, et al., J Pediatr Hematol Oncol 2015; 37: e 234-7.
　〔結果〕
　（ゲノム解析）
　まず、上記の最初の事例（ＵＰＮ１）における潜在的なゲノム変化を同定するために全ゲノムのシークエンシングを行い、ＦＩＳＨ及びＰＣＲで確認したところ、ＴＢＬ１ＸＲ１とＲＡＲＢとの間のフレーム内融合を同定することができた（図１のＥ及びＦ）。ＲＡＲＡのパラログであるＲＡＲＢはレチノイン酸レセプターβをコードしており、ＲＡＲＡもＲＡＲＢも核内レセプタースーパーファミリーのメンバーである。また、ＴＢＬ１ＸＲ１はＡＰＬにおけるＲＡＲＡのパートナー遺伝子として知られていた。そこで、さらに、全トランスクリプトームのシークエンシング、ＲＴ－ＰＣＲ及びＦＩＳＨを用いて、サンプルの入手性に応じて、ＲＡＲＡ陰性ＡＰＬと診断されたさらなる４例においてＲＡＲＢの変化について調べた（表１及び表２）。その結果、特に、２例では、ＵＰＮ１と同一の切断位置によるＴＢＬ１ＸＲ１－ＲＡＲＢ融合を有しており、１例では、ＦＩＳＨによって検出されたＲＡＲＢの再配列を有していたが、この１例のパートナー遺伝子は決定することができなかった。合計で５例中４例（８０％）のＲＡＲＡ陰性ＡＰＬの例がＲＡＲＢ転座を有していた。 〔result〕
(Genome analysis)
First, whole genome sequencing to identify potential genomic alterations in the first case above (UPN1), and by FISH and PCR confirmation, identify an in-frame fusion between TBL1XR1 and RARB (E and F in FIG. 1). RARB, a paralog of RARA, encodes retinoic acid receptor β, and both RARA and RARB are members of the nuclear receptor superfamily. TBL1XR1 was also known as a partner gene of RARA in APL. Thus, further, according to the availability of samples, further transcriptome sequencing, RT-PCR and FISH were used to examine changes in RARB in 4 additional cases diagnosed as RARA negative APL (Table 1 and Table 2). As a result, in particular, two cases had TBL1XR1-RARB fusion with the same cleavage position as UPN1, and one case had a rearrangement of RARB detected by FISH, but this one example The partner gene could not be determined. A total of 4 (80%) RARA negative APL cases had RARB translocations in total.
　以前の報告では、ＰＭＬ－ＲＡＲＡ単独では白血病を発症するのに不十分であり、ＪＡＫ２変異（Wartman LD, Larson DE, Xiang Z, et al., The Journal of clinical investigation 2011;121:1445-55）及びＦＬＴ３－ＩＴＤ（Madan V, Shyamsunder P, Han L, et al., Leukemia 2016;30:2430）などのさらなる事象を必要とすることが示された。それゆえ、ＲＡＲＢ転座以外の潜在的なゲノム変化を調べるために、サンプルが十分に入手可能な２例（ＵＰＮ１及びＵＰＮ２）を用いて全エクソームのシークエンシングを行った。その結果、反復変異を見つけることはできなかった（表３）が、１例では、ＲＡＲＢと融合していないもう一方のアレルにおいてＴＢＬ１ＸＲ１の体細胞変異を有していることがわかった。 In previous reports, PML-RARA alone was not sufficient to develop leukemia and JAK2 mutations (Wartman LD, Larson DE, Xiang Z, et al., The Journal of clinical investigation 2011; 121: 1445-55) And FLT3-ITD (Madan V, Shyams under P, Han L, et al., Leukemia 2016; 30: 2430) have been shown to require additional events. Therefore, in order to investigate potential genomic changes other than the RARB translocation, sequencing of whole exome was performed using two cases (UPN1 and UPN2) for which samples were sufficiently available. As a result, no repeat mutation was found (Table 3), but it was found that in one case, the other allele not fused to RARB had a TBL1XR1 somatic mutation.
　（レチノイン酸経路に対するＴＢＬ１ＸＲ１－ＲＡＲＢの影響）
　反復ＴＢＬ１ＸＲ１－ＲＡＲＢ融合は、ＴＢＬ１ＸＲ１－ＲＡＲＡ及びＰＭＬ－ＲＡＲＡと類似の構造を有していた（図２のＡ）（Chen Y, Li S, Zhou C, et al., Blood 2014;124:936-45）。そこで、ＡＰＬの病因に対するこの新規な融合の機能的影響を調べるために、ＴＢＬ１ＸＲ１－ＲＡＲＢ、ＰＭＬ－ＲＡＲＡ、ＲＡＲＢ及びＲＡＲＡを２９３Ｔ細胞株に形質導入した。ＲＡＲＡ／ＲＡＲＢは、レチノイン酸によって活性化するレチノイン酸経路を調節する転写因子である。ＴＢＬ１ＸＲ１－ＲＡＲＢの転写活性はＲＡＲＢと比べて小さく、この融合は、野生型ＲＡＲＢに対して優性な負の影響を有しており、ＡＴＲＡに対する応答におけるレチノイン酸経路の活性化を抑制した（図２のＢ）。さらに、ＴＢＬ１ＸＲ１－ＲＡＲＢ融合は、野生型ＲＡＲＡに対して優性な負の影響を有していた（図２のＣ）。 (Influence of TBL1XR1-RARB on retinoic acid pathway)
The repeated TBL1XR1-RARB fusion had a similar structure to TBL1XR1-RARA and PML-RARA (A in FIG. 2) (Chen Y, Li S, Zhou C, et al., Blood 2014; 124: 936- 45). Thus, to investigate the functional impact of this novel fusion on the pathogenesis of APL, TBL1XR1-RARB, PML-RARA, RARB and RARA were transduced into 293T cell lines. RARA / RARB is a transcription factor that regulates the retinoic acid pathway activated by retinoic acid. The transcriptional activity of TBL1XR1-RARB is smaller compared to RARB, and this fusion has a dominant negative effect on wild-type RARB and suppressed the activation of the retinoic acid pathway in response to ATRA (FIG. 2) B). In addition, the TBL1XR1-RARB fusion had a dominant negative effect on wild-type RARA (FIG. 2C).
　また、野生型ＲＡＲＢはＣＤ１１ｂの発現の増加を伴ってＵ９３７細胞株を分化させたが、ＴＢＬ１ＸＲ１－ＲＡＲＢはＵ９３７細胞株を分化させなかった（図２のＤ）。ＡＴＲＡによって、ＴＢＬ１ＸＲ１－ＲＡＲＢを形質導入したＵ９３７細胞株は分化したが、その効果はＰＭＬ－ＲＡＲＡのものよりもかなり少なかった。 In addition, wild type RARB differentiated the U937 cell line with an increase in expression of CD11b, but TBL1XR1-RARB did not differentiate the U937 cell line (FIG. 2D). Although ATRA differentiated the T937XR1-RARB transduced U937 cell line, the effect was much less than that of PML-RARA.
　ＡＰＬにおける全ての報告されたＲＡＲＡ融合は、ホモ二量体を形成する能力を共通の特徴として有している。ＴＢＬ１ＸＲ１のlissencephaly type-1-like homology motif（ＬｉｓＨ）ドメインはホモ二量体を形成する能力を有しているので、ＴＢＬ１ＸＲ１－ＲＡＲＢ融合タンパク質の二量体化能を調べた。ＰＭＬ－ＲＡＲＡ及びＴＢＬ１ＸＲ１－ＲＡＲＡ（Chen Y, Li S, Zhou C, et al., Blood 2014;124:936-45）と同様に、ＭＹＣタグ付きＴＢＬ１ＸＲ１－ＲＡＲＢは、ＨＡタグ付き融合タンパク質と免疫共沈殿することができたが、野生型ＲＡＲＢではできなかった（図２のＥ）。ＴＢＬ１ＸＲ１－ＲＡＲＢのホモ二量体化は治療量のＡＴＲＡによって弱められたが、その効果は部分的であった。ＡＴＲＡ及びタミバロテン（合成レチノイン酸）はＰＭＬ－ＲＡＲＡに対して強大な効果を有していたが、ＴＢＬ１ＸＲ１－ＲＡＲＢに対してはタミバロテンの効果も不十分であった。ＡＴＯは、ＴＢＬ１ＸＲ１－ＲＡＲＢに対してもちろん効果がない（図２のＥ）。これは、ＡＴＯが、多量化を妨害してＰＭＬ－ＲＡＲＡの分解を誘発するＰＭＬ部分を標的にするからである。 All reported RARA fusions in APL share the ability to form homodimers as a common feature. Since the lissencephaly type-1-like homology motif (LisH) domain of TBL1XR1 has the ability to form a homodimer, the dimerization ability of TBL1XR1-RARB fusion protein was examined. Similar to PML-RARA and TBL1XR1-RARA (Chen Y, Li S, Zhou C, et al., Blood 2014; 124: 936-45), MYC-tagged TBL1XR1-RARB is in immune co-existence with HA-tagged fusion proteins It was possible to precipitate but not in wild type RARB (FIG. 2E). Although homodimerization of TBL1XR1-RARB was attenuated by the therapeutic amount of ATRA, the effect was partial. ATRA and tamibarotene (synthetic retinoic acid) had a strong effect on PML-RARA, but the effect of tamibarotene on TBL1XR1-RARB was also insufficient. Of course, ATO has no effect on TBL1XR1-RARB (FIG. 2E). This is because ATO targets PML moieties that interfere with multimerization and induce PML-RARA degradation.
　ＴＢＬ１ＸＲ１－ＲＡＲＢによるＵ９３７細胞株の分化の阻害、及びＡＴＲＡに対する応答による部分的な好中球への分化を、形態学的変化によって確認した（図２のＦ）。 Inhibition of differentiation of the U937 cell line by TBL1XR1-RARB and partial differentiation into neutrophils in response to ATRA was confirmed by morphological changes (FIG. 2F).
　（細胞分化及び増殖に対すルＴＢＬ１ＸＲ１－ＲＡＲＢの影響）
　次いで、細胞分化及び増殖に対するこの新規な融合の影響をex vivoで評価した。ＴＢＬ１ＸＲ１－ＲＡＲＢ、ＲＡＲＢ及びＰＭＬ－ＲＡＲＡをネズミの骨髄に形質導入したところ、colony replating assayによって、ＴＢＬ１ＸＲ１－ＲＡＲＢがreplating能（野生型ＲＡＲＢでは観察されなかった）を向上できることが実証された（図３のＡ）。さらに、ヒト臍帯血の分化が、ＴＢＬ１ＸＲ１－ＲＡＲＢによって阻害され、ＰＭＬ－ＲＡＲＡ（形態学的特徴の変化によっても確認された）でも阻害された（図３のＢ））。
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
 (Influence of TBL1XR1-RARB on cell differentiation and proliferation)
The effect of this new fusion on cell differentiation and proliferation was then assessed ex vivo. Transfection of murine bone marrow with TBL1XR1-RARB, RARB and PML-RARA demonstrated that the colony replating assay demonstrated that TBL1XR1-RARB could improve the ability to replicate (which was not observed in wild-type RARB) (FIG. 3). A). Furthermore, human cord blood differentiation was inhibited by TBL1XR1-RARB and also by PML-RARA (also confirmed by changes in morphological characteristics) (FIG. 3B)).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
 　（ＴＢＬ１ＸＲ１－ＲＡＲＢに対する各種レチノイド投与の影響）
　ＴＢＬ１ＸＲ１－ＲＡＲＢ又はＰＭＬ－ＲＡＲＡを臍帯血細胞に形質導入した。
Figure JPOXMLDOC01-appb-T000003
 (Influence of administration of various retinoids on TBL1XR1-RARB)
Cord blood cells were transduced with TBL1XR1-RARB or PML-RARA.
　３７℃で１０日間幹細胞培養を行った後、各種のレチノイド（ＡＴＲＡ、Ａｍ８０、アシトレチン（Acitretin）、アリトレチノイン(Alitretinoin)、ベキサロテン（Bexarotene）、イソトレチノイン（Isotretinoin）又はタザロテン（Tazarotene））を投与し、さらに７日間培養し、骨髄細胞を得た。骨髄細胞にそれぞれ前回投与したものと同じ種類のレチノイドを投与し、さらに７日間培養した。培養開始から２４日目にＧＦＰ陽性細胞率及びＣＤ６６高発現細胞率を算出した。 After stem cell culture at 37 ° C. for 10 days, various retinoids (ATRA, Am80, acitretin), alitretinoin (Alitretinoin), bexarotene (Bexarotene), isotretinoin (Isotretinoin) or tazarotene (Tazarotene) are administered. The cells were further cultured for 7 days to obtain bone marrow cells. The bone marrow cells were administered with the same type of retinoid as that previously administered, and cultured for another 7 days. The percentage of GFP-positive cells and the percentage of cells expressing high CD66 were calculated 24 days after the start of culture.
　結果を図４に示す。図４は、ＰＭＬ－ＲＡＲＡ及びＴＢＬ１ＸＲ１－ＲＡＲＢに対する各種のレチノイド添加の影響を示す図である。図４の（Ａ）は、培養２４日目のＧＦＰ陽性細胞率を示す。コントロールとしてＤＭＳＯを添加した細胞の測定値を１としたときの各レチノイド添加サンプルの値を比較した。図４の（Ｂ）は、培養２４日目のＣＤ６６高発現細胞の割合（％）を示す。 The results are shown in FIG. FIG. 4 shows the effect of various retinoid additions on PML-RARA and TBL1XR1-RARB. (A) of FIG. 4 shows the percentage of GFP-positive cells on day 24 of culture. The value of each retinoid addition sample when the measured value of the cell which added DMSO as control was set to 1 was compared. (B) of FIG. 4 shows the percentage (%) of CD66 high expressing cells on day 24 of culture.
　その結果、ＲＡＲＢを形質導入した細胞において、イソトレチノインを添加した場合にＲＡＲＢ非導入細胞と比較して、ＧＦＰ陽性細胞率が低くなり（図４の（Ａ））、一方でＣＤ６６の発現量は高くなることがわかった（図４の（Ｂ））。以上より、イソトレチノインは細胞中のＴＢＬ１ＸＲ１－ＲＡＲＢ発現を抑制し、骨髄細胞増殖を正常化させるのに有効であることがわかった。 As a result, in cells transduced with RARB, the percentage of GFP-positive cells is lower when isotretinoin is added as compared to non-RARB cells (FIG. 4 (A)), while the expression level of CD66 is It turned out that it becomes high ((B) of FIG. 4). From the above, it was found that isotretinoin was effective in suppressing TBL1XR1-RARB expression in cells and normalizing bone marrow cell proliferation.
　以上のことから、種々のレチノイドのうち、特にイソトレチノインがＡＰＬの治療に有効である可能性があることが示唆された。 From the above, it was suggested that among various retinoids, isotretinoin, in particular, may be effective for the treatment of APL.
　本発明は、例えば、ＲＡＲＡが関与していないと考えられるＡＰＬの発症メカニズムの解明や、その治療法や治療薬の開発等に利用をすることができる。 The present invention can be used, for example, for elucidating an onset mechanism of APL in which RARA is considered not to be involved, and for developing a therapeutic method and a therapeutic agent thereof.

Claims (13)

  1. 　以下の１）～５）の何れかに記載されるポリヌクレオチド：
    １）ＲＡＲＢ（レチノイン酸受容体‐β）の一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
    ２）相互転座によって生じた、ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドをコードするポリヌクレオチド；
    ３）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
    ４）ＲＡＲＢの一部又は全長とＴＢＬ１ＸＲ１の一部又は全長との融合ポリペプチドであって、配列番号１のアミノ酸配列と８０％以上の配列同一性を有するアミノ酸配列で示されるポリペプチドをコードするポリヌクレオチド；
    ５）転座によって生じた、変異ＲＡＲＢをコードするポリヌクレオチド。     The polynucleotide described in any one of the following 1) to 5):
    1) A polynucleotide encoding a fusion polypeptide of a portion or the entire length of RARB (retinoic acid receptor-β) and a portion or the entire length of TBL1XR1;
    2) A polynucleotide encoding a fusion polypeptide of a part or full length of RARB and a part or full length of TBL1XR1 generated by mutual translocation;
    3) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1, which is a polynucleotide encoding the polypeptide represented by the amino acid sequence of SEQ ID NO: 1;
    4) A fusion polypeptide of a partial or full length of RARB and a partial or full length of TBL1XR1 which encodes a polypeptide represented by an amino acid sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 Polynucleotide;
    5) A polynucleotide encoding a mutated RARB produced by translocation.
  2. 　請求項１に示す何れかのポリヌクレオチドによってコードされているポリペプチド。 A polypeptide encoded by any of the polynucleotides of claim 1.
  3. 　対象より取得した試料における、請求項１に記載のポリヌクレオチド、及び／又は、請求項２に記載のポリペプチドを検出する検出工程を含む、検査方法。 A test method comprising a detection step of detecting the polynucleotide according to claim 1 and / or the polypeptide according to claim 2 in a sample obtained from a subject.
  4. 　上記検出工程の検出結果に基づいて、上記対象の分類を行う、請求項３に記載の検査方法。 The inspection method according to claim 3, wherein the classification of the object is performed based on the detection result of the detection step.
  5. 　上記対象の分類として、上記試料において、請求項１に記載のポリヌクレオチド、及び／又は、請求項２に記載のポリペプチドが検出される群Ａと、検出されない群Ｂとに分類する、請求項４に記載の検査方法。 The above object is classified into Group A in which the polynucleotide of Claim 1 and / or the polypeptide of Claim 2 is detected and Group B in which the polynucleotide of Claim 2 is not detected in the sample. The inspection method as described in 4.
  6. 　ＲＡＲＡ（レチノイン酸受容体‐α）の一部又は全長を含む融合ポリペプチド、及び／又は、当該融合ポリペプチドをコードするポリヌクレオチドが、上記群Ａでは上記試料において検出されない、請求項５に記載の検査方法。 6. A fusion polypeptide comprising part or the entire length of RARA (retinoic acid receptor-α) and / or a polynucleotide encoding the fusion polypeptide is not detected in said sample in said group A Inspection method.
  7. 　上記試料は血液又は骨髄液である、請求項３～６の何れか一項に記載の検査方法。 The test method according to any one of claims 3 to 6, wherein the sample is blood or bone marrow fluid.
  8. 　急性前骨髄性白血病（ＡＰＬ：Acute Promyelocytic Leukemia）に対する治療法を選択する方法であって、
    　請求項５～７の何れか一項に記載の検査方法によって分類される上記群Ａに属する対象に対して、通常よりも多量の、オールトランス型レチノイン酸（ＡＴＲＡ）を投与するか、ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法を選択する、方法。     A method of selecting a treatment for acute promyelocytic leukemia (APL) comprising
    An all-trans retinoic acid (ATRA) is administered to a subject belonging to the above-mentioned group A classified by the test method according to any one of claims 5 to 7 in a larger amount than usual, And / or a method of selecting a treatment other than ATO administration.
  9. 上記ＡＴＲＡ及び／又はＡＴＯ投与以外の治療法はイソトレチノインを投与する治療法である、請求項８に記載の方法。 9. The method of claim 8, wherein said treatment other than ATRA and / or ATO administration is a treatment wherein isotretinoin is administered.
  10. 　請求項１に記載のポリヌクレオチドが発現可能に導入されている細胞。 A cell in which the polynucleotide according to claim 1 is introduced so as to be expressible.
  11. 　請求項１に記載のポリヌクレオチド、請求項２に記載のポリペプチド、又は、請求項９に記載の細胞を用いて、急性前骨髄性白血病に対する有効性を評価する、評価方法。 An evaluation method for evaluating the efficacy against acute promyelocytic leukemia using the polynucleotide according to claim 1, the polypeptide according to claim 2, or the cells according to claim 9.
  12. 　請求項１に記載のポリヌクレオチドを検出するプライマー及び／又はプローブを含むか、請求項２に記載のポリペプチドを検出するプローブを含んでいる、キット。 A kit comprising a primer and / or a probe for detecting the polynucleotide of claim 1 or a probe for detecting the polypeptide of claim 2.
  13. 　イソトレチノインを有効成分として含む、ＲＡＲＡが関与していない急性前骨髄性白血病の治療薬。 A therapeutic agent for acute promyelocytic leukemia in which RARA is not involved, which comprises isotretinoin as an active ingredient.
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* Cited by examiner, † Cited by third party
Title
FREY J. R. ET AL.: "Antiproliferative activity of retinoids, interferon a and their combination in five human transformed cell lines", CANCER LETTERS, vol. 57, 1991, pages 223 - 227, XP026183387, ISSN: 0304-3835, DOI: doi:10.1016/0304-3835(91)90161-A *
KIYOI, HITOSHI: "(non-official translation) Treatment of Acute promyelocytic leukemia", THE JOURNAL OF THE JAPANESE SOCIETY OF INTERNAL MEDICINE, vol. 102, no. 7, pages 1705 - 1711, ISSN: 0021-5384 *
OSUMI T. ET AL.: "Recurrent RARB Translocations in Acute Promyelocytic Leukemia Lacking RARA Translocation", CANCER RESEARCH, vol. 78, no. 16, 19 June 2018 (2018-06-19), pages 4452 - 4458, XP055613653, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-18-0840 *

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