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WO2022124342A1 - Agent de traitement anticancéreux, et procédé de criblage pour son principe actif - Google Patents

Agent de traitement anticancéreux, et procédé de criblage pour son principe actif Download PDF

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WO2022124342A1
WO2022124342A1 PCT/JP2021/045165 JP2021045165W WO2022124342A1 WO 2022124342 A1 WO2022124342 A1 WO 2022124342A1 JP 2021045165 W JP2021045165 W JP 2021045165W WO 2022124342 A1 WO2022124342 A1 WO 2022124342A1
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cdkal1
cells
translation
gene
rna
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篤史 藤村
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Okayama University NUC
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to an agent for treating cancer and a method for screening an active ingredient thereof.
  • Gene expression in mammalian cells consists of two processes: transcription from DNA to messenger RNA (hereinafter sometimes referred to as "mRNA") and translation into which a protein is synthesized using the transcript mRNA as a template. ..
  • mRNA messenger RNA
  • mRNA messenger RNA
  • Cancer stem cells are cancer cells existing in cancer tissue that show stem cell properties, and an extremely small number of cancer stem cells present in cancer tissue cause proliferation, metastasis, recurrence, etc. of the cancer tissue. It is believed to be the cause. If it is possible to suppress the growth of cancer stem cells or lose the stem cell nature of cancer stem cells by inhibiting translation in cancer cells or cancer stem cells, it is expected to be applied as a therapeutic drug for cancer. However, as far as the present inventors know, there is no known drug that exerts a therapeutic effect by selectively inhibiting translation in cancer cells or cancer stem cells.
  • CDKAL1 Cdk5 regulatory subunit assisted protein 1-like 1 selectively recognizes tRNALys (UUU), which is a tRNA corresponding to the lysine codon AAA and AAG, and has a function of thiomethylating the 37th adenine of the tRNA. It is a tRNA modifying enzyme having (Non-Patent Document 1). Mutations in the gene encoding CDKAL1 are known to be associated with decreased insulin responsiveness and increased risk of developing type 2 diabetes (Non-Patent Document 2), and CDKAL1 has been widely studied in the field of diabetes research. Has been done.
  • Non-Patent Document 3
  • the relationship between CDKAL1 and cancer cells is unknown.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and targets a cancer cell or a cancer stem cell-specific translation, or a gene expressed by such translation.
  • a substance for cancer treatment which can suppress the proliferation of these cells or lose the stem cell property of cancer stem cells, or a substance which can be suitably used as an active ingredient of such an agent. It is an object to provide a screening method for the above.
  • the present inventors translate CDKAL1 into genes in cancer stem cells, and more specifically, to form translation initiation factor complexes on rough endoplasmic reticulum. Found to be involved in.
  • CDKAL1 has only been reported to be involved in the development of type 2 diabetes by modifying lysine tRNA, and CDKAL1 is involved in the translation of mRNA into protein in cells and in cancer stem cells.
  • the finding that it is done was a completely unexpected finding for the present inventors. Therefore, as a result of further diligent research efforts, the present inventors focused on the role of genes translated by the translation mechanism involved in CDKAL1 in cancer stem cells, and as a result, surprisingly, cancer stem cells.
  • the present invention solves the above-mentioned problems by providing an agent for treating cancer, which comprises, as an active ingredient, a component in which CDKAL1 suppresses the expression of a gene involved in its translation.
  • the translation mechanism in which CDKAL1 is involved is specifically activated in cancer cells, particularly cancer stem cells, and the expression of genes translated through the translation mechanism. Plays an important role in maintaining the stem cell nature of cancer stem cells, maintaining their self-renewal ability, and maintaining their tumorigenicity. Therefore, by suppressing the expression of the gene involved in the translation of CDKAL1, the self-renewal ability or tumor-forming ability of cancer cells can be suppressed, and thus an antitumor effect can be obtained.
  • the agent according to one aspect of the present invention is any stage of the gene expression process leading from DNA to protein production, as long as CDKAL1 can selectively suppress the expression of the gene involved in its translation. Alternatively, it may act on any molecule to suppress gene expression. Suppression of the expression of a gene in which CDKAL1 is involved in its translation is, for example, suppression of the expression of CDKAL1, reduction of mRNA in which CDKAL1 is a transcript of the gene involved in its translation, or from mRNA to protein. It can be achieved by inhibiting translation.
  • the present invention solves the above-mentioned problems by providing a method for screening a substance that inhibits translation in which CDKAL1 is involved.
  • the screening method according to the invention uses the following nucleic acid constructs; The first RNA sequence encoding the reporter protein and On the 5'end side, a second RNA sequence containing an RNA sequence represented by the following formula 1 and / or an RNA sequence represented by the following formula 2 and Nucleic acid construct encoding an RNA construct with (Equation 1) 5'-GGCGGGCGGGCGCGCGGC-3'(In the equation, the first G may be A, the second G may be C, the third C may be A, and the fourth G may be A.
  • the fifth G may be C
  • the sixth C may be A
  • the seventh G may be A or C
  • the eighth G may be A
  • the ninth C may be U or A
  • the 10th G may be A or U
  • the 11th G may be C
  • the 12th C may be A
  • the 13th G may be U or C or A
  • the 14th G may be U or A.
  • Or C, and the fifteenth C may be G, A, or U.
  • the 5th C may be U
  • the 6th C may be A or U
  • the 7th G may be U
  • the 8th C may be U
  • the 9th C may be G.
  • the 10th G can be U
  • the 11th C can be G
  • the 12th C can be U
  • the 13th G can be U
  • the 14th C can be U
  • the 15th. C may be G.
  • the mRNA translated by the translation mechanism in which CDKAL1 is involved has an RNA sequence characteristic of the untranslated region on the 5'end side thereof, in more detail.
  • the reporter protein reflecting the activity of the translation mechanism in which CDKAL1 is involved. Can be obtained. This makes it possible to easily and quickly screen substances that suppress or activate the translation mechanism in which CDKAL1 is involved.
  • the screening method according to the present invention is: (1) A step of introducing the nucleic acid construct into cells, (2) A step of contacting the cells into which the nucleic acid construct has been introduced with a solution containing a test substance or a solution not containing the test substance. (3) A step of measuring the intensity of a signal derived from the reporter protein in the cells contacted with the solution containing the test substance, and (4) A step of comparing the measured intensity of the signal with the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing no test substance. including.
  • a substance that inhibits the translation of the mRNA of the gene involved in the translation of CDKAL1 into a protein can be efficiently obtained.
  • the agent according to one aspect of the present invention is translated by involving a specific translation mechanism in cancer cells or cancer stem cells, and is used for maintaining the tumor-forming ability or self-renewal ability of cancer stem cells.
  • a specific translation mechanism in cancer cells or cancer stem cells By suppressing the expression of the genes involved, the growth of cancer stem cells can be suppressed.
  • the screening method according to another aspect of the present invention a substance that inhibits a translation mechanism specifically activated in cancer cells or cancer stem cells can be efficiently screened.
  • FIG. 3 is a microscopic image showing the expression state of stem cell markers (Vimentin, MSI1, Sox2, Nestin) or differentiation markers into nerve cells (SYS, MAP2) in malignant brain tumor cells JK2 infected with a lentiviral vector expressing shCDKAL1.
  • stem cell markers Vimentin, MSI1, Sox2, Nestin
  • SYS, MAP2 nerve cells
  • (A) It is a figure which shows the decrease in the sphere forming ability and the knockdown of CDKAL1 in human malignant melanoma cells A2058, SK-Mel-28, HMV-II infected with the lentiviral vector expressing shCDKAL1; (B). It is a figure which shows the decrease of the colony formation ability in A2058, SK-Mel-28, HMV-II which knocked down CDKAL1; (C) with the malignant melanoma stem cell marker in A2058, SK-Mel-28 which knocked down CDKAL1. It is a figure which shows the attenuation of the expression of a certain HMV1 and CD44.
  • (C) is the figure which shows the attenuation of the expression of ALDH1 and CD44 which are gastric cancer stem cell markers in NUGC3, HGC27 which knocked down CDKAL1.
  • (A) It is a figure showing the attenuation of the expression of SOX2, CD133, POU3F2, OLIG2, and CD44, which are malignant brain tumor stem cell markers in human malignant brain tumor stem cells MGG4, MGG8, and MGG18 infected with a lentiviral vector expressing shCDKAL1;
  • B) It is a figure which shows the decrease of the sphere formation ability in MGG4, MGG8, MGG18 which knocked down CDKAL1.
  • FIG. 1 It is a figure which shows the change of the tumor size with time in the mouse which was inoculated with the rhabdomyosarcoma cell RMS-YM cell which was infected with the lentiviral vector expressing shSALL2.
  • A A micrograph showing the morphology of Control-C2C12 cells prepared as a model of normal cells and HRas / skip53-C2C12 cells prepared as a model of malignant tumor cells, and a vector used for preparing a model of malignant tumor cells. It is a diagram showing the structure;
  • FIG. 1 is a diagram showing the primary structure and domain of CDKAL1;
  • B is a diagram showing the structure of wild-type CDKAL1 and the prepared CDKAL1 mutant;
  • C overexpressing wild-type CDKAL1 or CDKAL1 mutant.
  • the expression of CDKAL1 was knocked down by RNAi in the human rhombus myoma cell RD.
  • the composition of the protein contained in the cell lysate (input) obtained from the RD cells in which the expression of CDKAL1 was knocked down and the fraction (m7G bead precipitation) recovered from the cell lysate with m7GTP beads was analyzed by Western blotting.
  • CDKAL1 expression was knocked down by RNAi in human rhizome myoma cell RD overexpressing wild-type CDKAL1 or CDKAL1 variant. It is a figure which shows the result of having evaluated the sphere forming ability and the expression level of SALL2 protein by Western blotting in the RD cell which knocked down the expression of CDKAL1.
  • A is a diagram showing amino acids that may undergo post-translational modification in the amino acid sequences 1 to 202 from the amino end of CDKAL1 predicted by PhosphoSitePlus and ELM;
  • B wild-type CDKAL1 or CDKAL1 variants.
  • Reporter protein with or without knockdown of CDKAL1 in RD cells transfected with SALL2-5'UTR-firefly luciferase reporter plasmid, GAPDH-5'UTR-firefly luciferase reporter plasmid, or ACTB-5'UTR-firefly luciferase plasmid It is a figure which shows the change of the expression level (luciferase activity) of.
  • SALL2 is an RD cell transfected with the SALL2-5'UTR-firefly luciferase reporter plasmid
  • GAPDH is an RD cell transfected with the GAPDH-5'UTR-firefly luciferase reporter plasmid
  • ⁇ -ACTIN is ACTB-5'UTR.
  • (A) (i) DNA sequence encoding a first shRNA (shCDKAL1 # 1) targeting human CDKAL1, (ii) RNA sequence of shCDKAL1 # 1, (iii) RNA of siRNA that can result from shCDKAL1 # 1. Sequences; (B) (i) DNA sequence encoding a second shRNA targeting human CDKAL1 (shCDKAL1 # 2), (ii) RNA sequence of shCDKAL1 # 2, (iii) siRNA that can result from shCDKAL1 # 2. RNA sequence.
  • (A) (i) DNA sequence encoding a first shRNA (shSALL2 # 1) targeting human SALL2, (ii) RNA sequence of shRNA2 # 1, (iii) RNA of siRNA that can result from shSALL2 # 1. Sequences; (B) (i) DNA sequence encoding a second shRNA (shSALL2 # 2) targeting human SALL2, (ii) RNA sequence of shRNA2 # 2, (iii) siRNA that can result from shSALL2 # 2. RNA sequence.
  • the agent according to one aspect of the present invention is an agent for treating cancer, and more specifically, a component in which CDKAL1 (Cdk5 active substance subunit associated protein 1-like 1) suppresses the expression of a gene involved in its translation.
  • CDKAL1 CDK5 active substance subunit associated protein 1-like 1
  • CDKAL1 is involved in the formation of a translation initiation factor complex on the rough endoplasmic reticulum, which is one of the sites of protein synthesis, in cancer cells or cancer stem cells. It is based on the findings found by them.
  • gene expression is achieved by the process of transcription from DNA to mRNA, followed by translation from the transcript mRNA to protein.
  • translation from mRNA to protein is generally divided into three steps: "initiation” of translation, "elongation” of amino acid chain (protein) which is a translation product, and “termination” of translation.
  • the "initiation” of translation is the translation initiation factor complex formed by the binding of multiple eukaryotic translation Initiation Factor (eIF), and the 5'end 5'cap and 5'untranslation of the mRNA. It is said that it is started by the interaction of the regions.
  • eIF4A and eIF4G which are eukaryotic initiation factors, and eIF4E in cancer cells or cancer stem cells.
  • the translation initiation factor complex is formed by the action of CDKAL1.
  • CDKAL1 in the present specification means human CDKAL1 unless otherwise specified.
  • the expression of a gene means that the protein encoded by the gene is produced, and the suppression of the expression of the gene means reducing the production amount of the protein encoded by the gene, or reducing the production amount of the protein encoded by the gene. It means that the amount of production is reduced to zero.
  • the "treatment" of cancer includes not only the cure of the cancer but also the reduction of the cancer, the alleviation or improvement of the symptoms of the cancer, and the delay of the progression of the cancer. Is done.
  • the gene in which CDKAL1 is involved in the translation means a gene in which CDKAL1 is involved in the process of translating the mRNA of the gene into a protein, and more specifically, a gene in which CDKAL1 is involved in initiating translation of the gene. More specifically, it means a gene in which CDKAL1 is involved in the formation of a translation initiation factor complex involved in initiating translation of that gene.
  • the gene for which the agent according to the present invention suppresses its expression is not particularly limited as long as it is a gene in which CDKAL1 is involved in the translation of the gene in cancer cells or cancer stem cells, but is a preferred embodiment. In, it is preferable that it is a gene encoding a transcription factor. Examples of such genes include SALL2 gene, SP9 gene, IRF2BPL gene, ZNF276 gene, IFI35 gene, YAP1 gene, MIER1 gene, HOXA7 gene, PHF3 gene, LBX2 gene, KLF7 gene, HOXB6 gene, PLAG1 gene, ZNF484 gene.
  • the above genes are genes whose translations are regulated by the translation mechanism in which CDKAL1 is involved in cancer stem cells, and the transcription factors encoded by such genes are cancer stem cells.
  • the agent according to the present invention can exert a therapeutic effect on cancer by suppressing the expression of the gene subject to translation control by CDKAL1.
  • CDKAL1 Genes involved in the translation of CDKAL1 can be easily identified by those skilled in the art using genetic engineering techniques. For example, as shown in an experiment described later, in cells in which the expression of CDKAL1 was suppressed, the amount of each mRNA produced in the cell and the amount of each mRNA bound to a polysome (polyribosome), which is a place of translation, were measured. The measurement results are compared with the amount of each mRNA produced in the cell and the amount of each mRNA bound to the polysome when the expression of CDKAL1 is not suppressed in the same cell, and the amount of mRNA produced does not change. Nevertheless, it is only necessary to find a gene that reduces the amount of mRNA bound to polysomes (polyribosomes).
  • a gene whose intracellular mRNA production amount does not change due to suppression of CDKAL1 expression is a gene that is not affected at the transcription level by suppression of CDKAL1 expression.
  • Genes that reduce the amount of mRNA bound to polysomes, which are the sites of translation in cells, even though the amount of mRNA produced does not change, are genes that are regulated by CDKAL1 at the translation level, not at the transcription level, in other words.
  • CDKAL1 is a gene involved in its translation.
  • polysomes are granular organelles formed mainly on rough surface vesicles in eukaryotes, and have a structure in which a plurality of ribosomes are bound to one molecule of mRNA. Generally, it is considered that the greater the amount of mRNA bound to polysomes, the more actively translated mRNA.
  • the agent containing, as an active ingredient, a component in which CDKAL1 according to one aspect of the present invention suppresses the expression of a gene involved in its translation is not limited to those described below, and is not limited to those described below.
  • CDKAL1 may act on any stage or any molecule to suppress the expression of the gene involved in the translation.
  • the agent according to the present invention is, in one preferred embodiment thereof, an agent containing an ingredient that suppresses the expression of CDKAL1 as an active ingredient.
  • Such agents reduce the number of CDKAL1s that can participate in translation by suppressing the expression of CDKAL1, in other words, by reducing the amount of CDKAL1 produced in the cells, so that CDKAL1 is a gene involved in the translation. Can be suppressed.
  • knockout of CDKAL1 does not cause abnormalities in ontogeny, organ formation, etc. (Fan-Yan Wei et al. The Journal of Clinical Investment 2011, 121 (9).
  • the suppression of the expression of CDKAL1 is, for example, siRNA (small interfering RNA) for CDKAL1, shRNA ( It can be achieved by introducing short hairpin RNA), antisense nucleic acid, sgRNA (single guide RNA), or the like into a target cell.
  • the agent according to one aspect of the present invention may contain siRNA, shRNA, or sgRNA for CDKAL1 as an active ingredient in a preferred embodiment thereof.
  • siRNA means double-stranded RNA capable of knocking down a gene by RNA interference.
  • the number of base pairs constituting the double-stranded RNA is not particularly limited, but is, for example, 18 to 30 base pairs, 20 to 27 base pairs, and typically 21 to 23 base pairs of double-stranded RNA.
  • RISC RNA-induced silencing complex
  • siRNA is introduced into cells, it forms an RNA-protein complex called an RNA-induced silencing complex (RISC) with Argonaute protein, and forms a sequence complementary to the antisense strand of siRNA. Suppresses the expression of mRNA. That is, the siRNA for CDKAL1 can be a double-stranded RNA containing an RNA having a base sequence complementary to the mRNA of CDKAL1.
  • complementary means that the first base forms a classical Watson-Crick base pair or a non-Watson-Crick base pair with a second base and hydrogen bonds. Means that can be formed. Also, in the present specification, when two base sequences are “complementary", all consecutive bases in the first base sequence are complementary to the same number of consecutive bases in the second base sequence, that is, Not only when hydrogen bonds can be formed (this case may be referred to as “fully complementary"), but of all the bases in the first base sequence, for example, 70% or more, 80% or more, or 90. Includes the case where% or more of the bases can form hydrogen bonds with the bases of the second base sequence.
  • siRNA may contain RNA having a base sequence completely complementary to a part of mRNA which is a transcript of the CDKAL1 gene, or from a completely complementary base sequence. A few bases may contain RNA having a modified base sequence. Also, in one preferred embodiment, each 3'end of the sense strand and antisense strand constituting the siRNA may have an overhang of 2 to 5 nucleotides or modified nucleotides, for example. It may have two deoxy-thymidines (dTdT).
  • the siRNA for CDKAL1 can be appropriately designed by those skilled in the art based on the DNA sequence of CDKAL1 (A. Birmingham et al. Nature Protocols, 2007, 2, 2068-2078; E. Fakhr et al. Cancer Gene. Therapy, 2016, 23, 73-82.). Also known software (Yuki Naito et al. Nucleic Acids Research, Volume 32, Issue suppl_2, 1, July 2004, Pages W124-W129; Simone Sciabola et al. InvivoGen)) and the like may be used.
  • the DNA sequence of human CDKAL1 can be obtained from a public database, for example, the National Center for Biotechnology Information (NCBI), and the DNA sequence set forth in SEQ ID NO: 20 of the sequence listing. May be used.
  • the RNA sequence of siRNA for human CDKAL1 is, for example, the RNA sequence shown in FIG. 27A (SEQ ID NOs: 3 and 4), the RNA sequence shown in FIG. 27B (SEQ ID NOs: 7 and 8), or the arrangement.
  • the RNA sequences set forth in SEQ ID NOs: 41 and 42 in the column table can be used, but the sequence of siRNA for CDKAL1 that can be used in the present invention is not limited thereto.
  • any of the above siRNAs may be mutated such that a nucleotide residue is inserted, deleted or substituted.
  • the number of the mutations is not particularly limited, but may be, for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.
  • shRNA is a hairpin-type RNA used for knocking down a gene by RNA interference.
  • the hairpin structure of shRNA is cleaved in the cell to produce double-stranded RNA of about 21 to 23 base pairs.
  • the generated double-stranded RNA forms an RNA-protein complex called RISC with the Argonaute protein, as described for siRNA, and expresses an mRNA having a sequence complementary to the antisense strand of the double-stranded RNA.
  • the shRNA for CDKAL1 can be a hairpin-type RNA containing a base sequence complementary to the mRNA of CDKAL1.
  • the shRNA for human CDKAL1 can be appropriately designed by those skilled in the art based on the DNA sequence of human CDKAL1, and can also be designed using known software.
  • sequence of shRNA for human CDKAL1 for example, the RNA sequence shown in FIG. 27A (SEQ ID NO: 2) or the RNA sequence shown in FIG. 27B (SEQ ID NO: 6) can be used, but the present invention.
  • the sequence of shRNA for CDKAL1 that can be used in is not limited to these.
  • any of the above shRNAs may be mutated such that a nucleotide residue is inserted, deleted or substituted.
  • the number of the mutations is not particularly limited, but may be, for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.
  • Antisense nucleic acid is a single-stranded DNA or RNA having an action of inducing translational repression of the mRNA by hybridizing with the mRNA of the target gene. That is, the antisense nucleic acid for CDKAL1 can be a single-stranded DNA or RNA containing a base sequence complementary to the mRNA of CDKAL1.
  • the antisense nucleic acid for CDKAL1 can be appropriately designed by those skilled in the art based on the DNA sequence of CDKAL1 (J. HP Chan et al. Clinical and Experimental Pharmacology and Physiology, 2006, Vol. 33, Vol. 33, Vol. 540.). Further, known software (for example, Simone Sciabola et al.
  • the antisense nucleic acid may be one in which RNA or DNA complementary to the sequence is bound, and the antisense nucleic acid to which such complementary RNA or DNA is bound may be used.
  • it can be a hetero double-stranded nucleic acid (DNA / RNA) or a homo double-stranded nucleic acid (DNA / DNA) (K. Nishina et al. Nat. Commun. 2015, 6, 7769; Y. Asami et al. Molecular. Therapy, 2021, 29, 838-847.).
  • sgRNA is a single-stranded RNA having a sequence complementary to the target DNA, and when sgRNA is introduced into a cell together with a specific endonuclease, the DNA double strand having a sequence complementary to sgRNA is cleaved.
  • the gene encoded by the DNA can be specifically knocked out.
  • the endonuclease is a Cas9 nuclease derived from Streptococcus pyogenes or a variant thereof.
  • the sgRNA can be appropriately designed by those skilled in the art based on the base sequence of the target gene.
  • RNA molecules such as siRNA, ThenRNA, antisense nucleic acid or sgRNA used in the present invention are intended to improve stability, gene expression inhibitory effect, cell introduction efficiency and the like.
  • it may be chemically modified.
  • the phosphate group of the RNA molecule may be replaced with a chemically modified phosphate group such as phosphorothioate, methylphosphonate, or phosphorodithionate, or the RNA molecule may be replaced with a chemically modified phosphate group.
  • a part of the constituent nucleic acid may be replaced with peptide nucleic acid (PNA).
  • a polymer such as polyethylene glycol, a peptide such as cholesterol or a cell-permeable peptide, a sugar or sugar chain such as GalNAc (N-Acetylgalactosamine), an antibody, an antibody fragment, an aptamer, etc. are bound to the 3'end or 5'end. It may be the one that has been used.
  • the agent according to the present invention comprises, in a preferred embodiment thereof, an expression vector expressing siRNA, shRNA, antisense nucleic acid or sgRNA in the cell instead of siRNA, ThenRNA, antisense nucleic acid or sgRNA.
  • expression vectors include plasmids, cosmids, phagemids, viral vectors and the like, and examples of viral vectors include lentivirus vectors, retroviral vectors, adenoviral vectors, adeno-associated virus vectors, Sendai virus vectors and the like. Viral vectors are exemplified, but not limited to these.
  • These expression vectors can be appropriately prepared by those skilled in the art (G. Sui et al., Proc. Natl. Acad. Sci. USA 2002, 99 (8), 5515-5520. .).
  • RNA molecules such as siRNA, shRNA or sgRNA can be introduced into cells by an appropriate method regardless of physical or chemical methods, depending on the type of cell to be introduced and the environment in which the cells are present. Then, an appropriate method may be selected. Examples of the physical introduction method include an electroporation method, a sonoporation method, and a microinjection method. On the other hand, as a chemical introduction method, in addition to the calcium phosphate method and the lipofection method using liposomes, cationic lipids, lipidoids, cationic polymers, membrane-permeable peptides, antibodies, antibody fragments, proteins, nanoparticles, and microparticles.
  • RNA molecule may be administered systemically in combination with an appropriate delivery means if necessary, or the tissue. May be administered topically by injection or application to.
  • the agent according to the present invention may be an agent containing a component in which CDKAL1 reduces the mRNA of a gene involved in its translation in a preferred embodiment thereof.
  • the agent containing a component in which CDKAL1 reduces the mRNA of the gene involved in its translation the expression of the gene can be suppressed.
  • the reduction of mRNA can be achieved by suppressing the production of mRNA or by degrading the produced mRNA.
  • the mRNA of the gene may not be produced by knocking out the gene that is transcribed to produce the mRNA in the target cell.
  • agents include, for example, shRNA, siRNA, antisense nucleic acid or sgRNA for the gene as an active ingredient.
  • the siRNA for the gene in which CDKAL1 is involved in its translation can be a double-stranded RNA comprising an RNA having a base sequence complementary to the mRNA that is the transcript of the gene.
  • the specific genes in which CDKAL1 is involved in its translation are as described above, but for example, in one preferred embodiment, the gene can be SALL2.
  • a person skilled in the art can appropriately design siRNA for a certain gene based on the DNA sequence of the gene.
  • Human SALL2 DNA sequences can also be obtained from public databases, such as the National Center for Biotechnology Information (NCBI). Specific examples of the siRNA sequence for human SALL2 include the RNA sequences set forth in FIG.
  • any of the above siRNAs may be mutated such that a nucleotide residue is inserted, deleted or substituted.
  • the number of the mutations is not particularly limited, but may be, for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.
  • the shRNA for the gene in which CDKAL1 is involved in its translation can be a hairpin-type RNA containing a base sequence complementary to mRNA which is a transcript of the gene.
  • the specific genes in which CDKAL1 is involved in its translation are as described above, but for example, in one preferred embodiment, the gene can be SALL2. That is, in one preferred embodiment, the shRNA for the gene in which CDKAL1 is involved in its translation can be shRNA for SALL2.
  • Specific examples of the shRNA sequence for human SALL2 include the RNA sequence shown in FIG. 28A (SEQ ID NO: 10) or the RNA sequence shown in FIG. 28B (SEQ ID NO: 14). The sequence of shRNA for SALL2 that can be used in is not limited to these.
  • any of the above shRNAs may be mutated such that a nucleotide residue is inserted, deleted or substituted.
  • the number of the mutations is not particularly limited, but may be, for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, and even more preferably 1.
  • siRNA siRNA, antisense nucleic acid, and sgRNA, other parts including the introduction method and preparation method thereof are as described above.
  • the agent according to the present invention may be an agent containing a component that inhibits the translation of the gene involved in the translation of CDKAL1 from mRNA to protein in a preferred embodiment thereof.
  • Inhibiting translation here means inhibiting the process of protein synthesis using mRNA, which is a transcript of a gene, as a template, and the translation is a translation involving CDKAL1, more specifically, CDKAL1.
  • the translation involved in the initiation When the above agent inhibits the translation involving CDKAL1, and more specifically, the translation of the mRNA initiated by the involvement of CDKAL1, the production amount of the protein corresponding to the mRNA is reduced. In other words, CDKAL1 suppresses the expression of genes involved in its translation.
  • the present inventors have been able to stabilize the translation initiation factor complex in which CDKAL1 is involved at positions 1 to 202 from the amino terminus containing the UPF domain of CDKAL1.
  • the amino acid sequence of is playing an important role.
  • the present inventors have made post-translational modifications in the amino acid sequences 1 to 202 from the amino terminus containing the UPF domain of CDKAL1, specifically, N-binding in the amino acid sequence.
  • Type glycosylation and phosphorylation by GSK3 (Glycogen synthesis kinase 3), more specifically, N-linked glycosylation of the 107th asparagine from the N-terminus of CDKAL1, and the 18th and 22nd from the N-terminus of CDKAL1.
  • GSK3 Glycogen synthesis kinase 3
  • N-linked glycosylation of the 107th asparagine from the N-terminus of CDKAL1 and the 18th and 22nd from the N-terminus of CDKAL1.
  • phosphorylation of the 153rd serine by GSK3 plays an important role in stabilizing the translational initiation factor complex in which CDKAL1 is involved, and according to the N-linked glycosylation inhibitor or GSK3 inhibitor, CDKAL1 It was found that the stabilizing effect of the translation initiation factor complex involving CDKAL1 was significantly reduced, and the translation involving CDKAL1 was inhibited.
  • the component that inhibits translation involving CDKAL1 can be an N-linked glycosylation inhibitor or a GSK3 inhibitor.
  • N-linked glycosylation inhibitors or GSK3 inhibitors there are no particular restrictions on the types of N-linked glycosylation inhibitors or GSK3 inhibitors that can be used, such as tunicamycin as an N-linked glycosylation inhibitor and BIO or CHIR-98014 as a GSK3 inhibitor. Can be used.
  • the component that inhibits the translation of the gene involved in its translation from mRNA to protein can be a component that specifically binds to CDKAL1.
  • Translation involving CDKAL1 requires the formation of a translation initiation factor complex involving CDKAL1, whereas a component that specifically binds to CDKAL1 inhibits the interaction between CDKAL1 and the translation initiation factor complex. This can inhibit translations involving CDKAL1.
  • the amino acid sequences 1 to 202 from the amino terminus containing the UPF domain of CDKAL1 are important for stabilizing the translation initiation factor complex in which CDKAL1 is involved. Playing a role. Therefore, the component that specifically binds to the amino acid sequence 1 to 202 from the amino terminus including the UPF domain of CDKAL1 inhibits the interaction between CDKAL1 and the translation initiation factor complex and suppresses the translation mechanism in which CDKAL1 is involved. Can be suitably used for this purpose.
  • Glycosylation and GSK3 phosphorylation of the 18th, 22nd, and 153rd serines from the N-terminus of CDKAL1 play important roles in stabilizing the translation initiation factor complex in which CDKAL1 is involved. Therefore, the amino acid sequence 1 to 202 from the amino terminus of CDKAL1 that specifically binds to the amino acid sequence containing one or more post-translational modifications of (1) to (4) below is the same as CDKAL1.
  • CDKAL1 It may be particularly preferably used to inhibit interaction with the translational initiation factor complex and suppress the translational mechanism involving CDKAL1: (1) Phosphorylation of the 18th serine from the N-terminal; (2) Phosphorylation of the 22nd serine from the N-terminal; (3) N-linked glycosylation of the 107th asparagine from the N-terminus; (4) Phosphorylation of the 153rd serine from the N-terminal.
  • the type of component that specifically binds to CDKAL1 is not particularly limited, but may be, for example, a peptide, an antibody, an antibody fragment, an aptamer, or the like. Those skilled in the art can prepare these substances by an appropriate method based on the disclosed contents of the present specification and known techniques.
  • peptide means a molecule in which natural or non-natural amino acids are dehydrated and condensed, and whether it is a linear peptide or a branched chain peptide. It may be a cyclic peptide.
  • the number of amino acids constituting the peptide is not particularly limited, but is typically 50 or less, preferably 40 or less, more preferably 30 or less, still more preferably 20 or less.
  • Peptides that specifically bind to CDKAL1 can be described by those skilled in the art using the phage display method (GP Smith, Science 1985, Vol. 228, Issue 4705, pp. 1315-1317.), Ribosome display method (L. C. Mattheakis et al. Proc. Natl. Acad.
  • a phage library presenting a peptide library having a random amino acid sequence is prepared, and a target protein or a group of phage that binds to a part of the target protein is selected from the phage library. ..
  • the phage library is contacted with the immobilized target protein to remove the phage group that did not bind to the target protein, and then the phage bound to the target protein is eluted, infected with Escherichia coli, and amplified.
  • phages having high binding property to the target protein can be obtained, in other words, the amino acid sequence of the peptide having high binding property to the target protein can be determined.
  • a part or all of the amino acid sequence of CDKAL1 can be used, but preferably a part or all of the amino acid sequence 1 to 202 from the amino end of CDKAL1, and more preferably the amino acid of CDKAL1.
  • a region which is a part or all of the amino acid sequence 1 to 202 from the terminal and contains 1 or 2 or more of the following (1) to (4) can be preferably used: (1) Phosphorylated N-terminal 18th serine; (2) Phosphorylated N-terminal 22nd serine; (3) N-linked glycosylated asparagine 107th from the N-terminus; (4) The 153rd serine from the phosphorylated N-terminal.
  • the term "antibody” refers to an immunoglobulin molecule that is naturally produced or produced using genetic recombination technology, and the "antibody fragment” is an antigen-binding molecule contained in the immunoglobulin molecule. Say a fragment.
  • the antibody may be any of a polyclonal antibody, a monoclonal antibody, and a recombinant antibody.
  • a polyclonal antibody refers to a set of a plurality of types of immunoglobulins that recognize and bind to different epitopes of the same antigen, while a monoclonal antibody refers to a clone group of a single immunoglobulin.
  • Recombinant antibody means an antibody produced by combining amino acid sequences of antibodies derived from different animals such as chimeric antibody and humanized antibody.
  • Antibody fragments include, for example, F (ab') 2 , F (ab) 2 , Fab', Fab, Fv, scFv and the like.
  • an antibody or antibody fragment that specifically binds to CDKAL1 can be prepared by those skilled in the art using known techniques.
  • polyclonal antibodies can be obtained from plasma of immune animals administered with an antigen and, if necessary, an adjuvant.
  • a monoclonal antibody can also be obtained by the hybridoma method. More specifically, antibody-producing cells (for example, B cells) were obtained from an immune animal to which an antigen and, if necessary, an adjuvant were administered, and the obtained antibody-producing cells were fused with myeloma or the like to prepare a hybridoma.
  • a monoclonal antibody can be obtained by purifying the antibody produced by the hybridoma.
  • the antigen used for immunization may be a part or all of the amino acid sequence of CDKAL1, but preferably a part or all of the amino acid sequence 1 to 202 from the amino end of CDKAL1, more preferably CDKAL1.
  • a region having an amino acid sequence including 1 or 2 or more of the following (1) to (4), which is a part or all of the amino acid sequence 1 to 202 from the amino end of the above, can be preferably used: (1) Phosphorylated N-terminal 18th serine; (2) Phosphorylated N-terminal 22nd serine; (3) N-linked glycosylated asparagine 107th from the N-terminus; (4) The 153rd serine from the phosphorylated N-terminal.
  • the method for producing an antibody or antibody fragment is not limited to the above, and may be produced using a gene recombination technique.
  • the monoclonal antibody produced by the hybridoma produced by the above procedure the gene encoding the heavy chain variable region or light chain variable region, heavy chain CDR, light chain CDR, etc. of the monoclonal antibody is cloned to obtain the gene.
  • the obtained vector is introduced into a host cell to transform the host cell, whereby a cell producing an antibody or an antibody fragment thereof can be obtained.
  • An antibody or an antibody fragment thereof can also be obtained by purifying the antibody produced by the cells.
  • Those skilled in the art can appropriately set the type of host cell, the type of vector, the culture conditions, etc. used for preparing the antibody or the antibody fragment thereof.
  • the term "aptamer” as used herein refers to a nucleic acid that specifically binds to a specific substance.
  • the aptamer for CDKAL1 can be produced by a person skilled in the art by an appropriate method.
  • Such a method includes, for example, the SELEX method (Systematic Evolution of Ligands by EXPonential evolution) (Tuerk, C .; Gold, L. Science 1990, 249, 505.).
  • SELEX method Systematic Evolution of Ligands by EXPonential evolution
  • a nucleic acid library having a random base sequence is prepared, and a target protein or a group of nucleic acids that bind to a part of the target protein is selected from the library.
  • the selected nucleic acid group is amplified by the PCR method to obtain a nucleic acid library.
  • a nucleic acid library By repeating the selection and amplification cycle several to several tens of times, it is possible to identify a sequence of a nucleic acid having a strong binding force to a target protein or a part of the target protein.
  • the component that inhibits translation involving CDKAL1 can be a small molecule compound.
  • a large number of small molecule compounds that inhibit translation involving CDKAL1 can be efficiently obtained by the screening method according to one aspect of the present invention, which will be described later.
  • such low molecular weight compounds include, for example, Go6983, tunicamycin, Ozanimod, Gramicidin, Lomitapide, Fenticonazole (Nitrate), Asenapine (hydrochloride), Proph.
  • the small molecule compound having an action of suppressing the translation mechanism in which CDKAL1 is involved can be suitably used as an active ingredient of an agent for treating cancer.
  • the component that inhibits translation involving CDKAL1 that can be used in the agent according to another aspect of the present invention may have an activity that inhibits translation involving CDKAL1, and the type thereof is not particularly limited. do not have. It may be a natural substance, a synthetic substance, an organic compound or an inorganic compound, a low molecular weight compound having a molecular weight of up to about 500, a medium molecular weight compound having a molecular weight of about 500 to 1000, or more. It may be a polymer compound having a molecular weight of.
  • it may be a linear or cyclic peptide, amino acid, protein, antibody, antibody fragment, nucleic acid, sugar, lipid, natural polymer, synthetic polymer, inorganic compound, organic compound, or a combination thereof. ..
  • a substance that inhibits translation in which CDKAL1 is involved can be easily obtained by the screening method according to one aspect of the present invention, which will be described later.
  • cancer there is no particular limitation on the types of cancer to which the agent according to one aspect of the present invention can be applied, and the translation mechanism in which CDKAL1 is involved in the cancer cells constituting the cancer to be treated, more specifically, CDKAL1
  • CDKAL1 basically any cancer can be used as long as the translation mechanism involved in its initiation is activated.
  • cancer refers to a hematopoietic malignant tumor such as leukemia, lymphoma, and sarcoma, and a malignant tumor (cancer or malignant tumor) originating from epithelial cells such as lung cancer, breast cancer, gastric cancer, and colon cancer.
  • Carcinoma) and malignant tumors (sarcoma or sarcoma) originating from non-epithelial cells such as osteosarcoma, chondrosarcoma, rhizome myoma, and smooth myoma) are included.
  • the cancer to be treated by the agent according to the present invention is a sarcoma or an epithelial malignant tumor, more preferably a rhabdomyosarcoma or a malignant brain tumor such as a glioma.
  • Glioblastoma includes glioblastoma (glioblastoma), and glioblastoma may be mesenchymal type or proneural type.
  • CDKAL1 by suppressing the translation mechanism involved in CDKAL1, it is possible to suppress the growth of a wide variety of cancers including rare cancers such as malignant brain tumors and rhabdomyosarcoma.
  • the agent according to the invention further comprises water, a buffer (eg, a phosphate buffer, a borate buffer, a citric acid buffer, a tartrate buffer, an acetate buffer, depending on its use.
  • a buffer eg, a phosphate buffer, a borate buffer, a citric acid buffer, a tartrate buffer, an acetate buffer, depending on its use.
  • Agents, amino acids, etc.), preservatives eg, quaternary ammonium salts such as benzalconium chloride, paraoxybenzoic acid esters such as methyl paraoxybenzoate, benzyl alcohol, sorbic acid and its salts, thimerosal, parabens, etc.
  • chelates eg, quaternary ammonium salts such as benzalconium chloride, paraoxybenzoic acid esters such as methyl paraoxybenzoate, benzyl alcohol, sorbic acid and its salts, thimerosal, paraben
  • Pharmaceutically acceptable 1 to be incorporated into agents eg, sodium edetate, citric acid, etc.
  • antioxidants eg, sodium hydrogen sulfite, sodium sulfite, sodium pyrosulfate, etc.
  • the agent according to the present invention can be provided as a pharmaceutical composition.
  • the administration route of the agent according to the present invention is not particularly limited, and an appropriate administration route may be selected according to the cancer tissue to be applied.
  • oral administration, sublingual administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, local administration and the like are exemplified, but not limited thereto.
  • the drug may be formulated in a dosage form suitable for the route of administration according to the route of administration.
  • Dosage forms suitable for oral administration include, for example, tablets, capsules, powders, granules, syrups, etc.
  • dosage forms suitable for parenteral administration include, for example, solution-type injections, suspensions, etc. Examples thereof include injections such as liquid injections and time-prepared injections.
  • the following nucleic acid constructs are provided: The first RNA sequence encoding the reporter protein and On the 5'end side, a second RNA sequence containing an RNA sequence represented by the following formula 1 and / or an RNA sequence represented by the following formula 2 and Nucleic acid construct encoding an RNA construct with (Equation 1) 5'-GGCGGGCGGGCGCGGC-3'(In the equation, the first G may be A, the second G may be C, the third C may be A, and the fourth G may be A.
  • the fifth G may be C, the sixth C may be A, the seventh G may be A or C, the eighth G may be A, the ninth C may be U or A, and so on.
  • the 10th G may be A or U
  • the 11th G may be C
  • the 12th C may be A
  • the 13th G may be U or C or A
  • the 14th G may be U or A
  • the fifteenth C may be G, A, or U.
  • (Equation 2) 5'-GCCGCCGCCGCCGCC-3'(In the equation, the first G may be U or C, the second C may be G, the third C may be U, and the fourth G may be U.
  • the 5th C may be U
  • the 6th C may be A or U
  • the 7th G may be U
  • the 8th C may be U
  • the 9th C may be G.
  • the 10th G can be U
  • the 11th C can be G
  • the 12th C can be U
  • the 13th G can be U
  • the 14th C can be U
  • the 15th. C may be G.
  • the mRNA translated by the translation mechanism involving CDKAL1 is an RNA sequence characteristic of the untranslated region on the 5'end side thereof, more specifically, cytosine (C). And an RNA sequence rich in guanine (G), more specifically, an RNA sequence represented by the above formula 1 or formula 2.
  • Cytosine and guanine-rich RNA sequences are known to form complex secondary structures, and translation factor complexes have such RNA sequences in the untranslated region on the 5'end to translate mRNAs. It is believed that involvement is needed. That is, the amount of the reporter protein encoded by the RNA construct in the cell reflects the activity of the translation mechanism in which CDKAL1 is involved in the cell. Therefore, the nucleic acid construct encoding the RNA construct can be suitably used, for example, for screening a substance having an activity of inhibiting or promoting a translation mechanism in which CDKAL1 is involved.
  • RNA sequences represented by the above formula 1 or 2 contained in the second RNA sequence of the RNA construct there is no particular limitation on the number of RNA sequences represented by the above formula 1 or 2 contained in the second RNA sequence of the RNA construct, but for example, 1 to 12, preferably 1 to 10 pieces. , More preferably 2 to 8, and even more preferably 4 to 8.
  • the upper limit (4) and the lower limit (8) are included unless otherwise specified. It means a numerical range.
  • the second RNA sequence of the RNA construct can be the RNA sequence of the 5'untranslated region of the mRNA of the gene in which CDKAL1 is involved in its translation.
  • an RNA sequence in the 5'untranslated region of the mRNA of the human SALL2 gene can be preferably used, but is not limited thereto.
  • the DNA sequence corresponding to the RNA sequence of the 5'untranslated region of the human SALL2 gene mRNA is as set forth in SEQ ID NO: 32 of the sequence listing.
  • the second RNA sequence possessed by the RNA construct is the RNA sequence of the 5'untranslated region of the mRNA of the gene involved in the translation of CDKAL1.
  • the RNA sequence contains an RNA sequence in which CDKAL1 is substantially the same as the RNA sequence of the 5'untranslated region of the mRNA of the gene involved in its translation.
  • an RNA sequence that is substantially the same as an RNA sequence is an RNA sequence having 80-100%, preferably 90-100%, more preferably 95-100% identity as compared to an RNA sequence. Means.
  • the identity of the RNA sequence can be appropriately determined by those skilled in the art based on a sequence analysis algorithm such as BLAST.
  • the RNA construct encoded by the nucleic acid construct according to one aspect of the present invention has a first RNA sequence encoding a reporter protein.
  • the reporter protein is not particularly limited in its type as long as it is a protein capable of directly or indirectly measuring a signal reflecting the amount of the present reporter protein, and can be used by the user.
  • Appropriate reporter proteins may be used depending on the measuring instrument. For example, when a luminescence photometer is available, luminescent enzyme proteins such as firefly luciferase, sea urchin luciferase, and marine copepod luciferase can be used as reporter proteins.
  • a blue fluorescent protein (BFP: Blue Fluorescent Protein)
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • RFP Red Fluorescent Protein
  • a fluorescent protein such as a protein (RFP: Red Fluorescent Protein) may be used as a reporter protein.
  • RFP Red Fluorescent Protein
  • a color-developing enzyme protein such as ⁇ -galactosidase can be used as a reporter protein.
  • the base sequence encoding the reporter protein can be obtained from a public database, for example, a database of the National Center for Biotechnology Information (NCBI).
  • the nucleic acid construct can typically be a DNA construct encoding the RNA construct, but may be an expression vector prepared by inserting the RNA construct or the DNA encoding the RNA construct.
  • expression vectors include plasmids, phages, cosmids, phagemids, viral vectors, and examples of virus vectors include lentivirus vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus.
  • Vectors and the like are exemplified.
  • the type of expression vector is not particularly limited, and a vector that can be expressed in a target cell and can produce the RNA construct may be appropriately selected.
  • the cells containing the nucleic acid construct described above can express the reporter protein depending on the activity of CDKAL1. Therefore, cells containing the nucleic acid construct can be suitably used, for example, for screening substances having an activity of inhibiting or promoting the translation mechanism in which CDKAL1 is involved. Thus, according to another aspect of the invention, a cell containing the nucleic acid construct is provided.
  • the cells containing the nucleic acid construct according to one aspect of the present invention are not particularly limited, but are preferably mammalian cells, more preferably human cells.
  • cells in which the translation mechanism in which CDKAL1 is involved are activated, for example, cancer cells are preferable, and in particular, cancer stem cells are more preferable.
  • cancer cells are preferable, and in particular, cancer stem cells are more preferable.
  • type of cancer cells or cancer stem cells but for example, malignant melanoma, liver cancer, prostate cancer, gastric cancer, malignant brain tumor, cancer cells derived from rhizome myoma, or cancer stem cells. It is more preferable that it is a cancer cell derived from a malignant brain tumor or a collateral myoma, or a cancer stem cell.
  • RD cells include, but are not limited to, RD cells, JK2 cells, and RMS-YM cells.
  • Whether or not the translation mechanism in which CDKAL1 is involved is activated in a certain cell can be confirmed by a person skilled in the art by an appropriate method. For example, according to the method described in Experiment 2 described later, the effect of the presence or absence of knockdown of CDKAL1 on the formation of the translation initiation factor complex may be analyzed by Western blotting.
  • the nucleic acid construct according to one aspect of the present invention or the cell containing the nucleic acid construct can be suitably used for screening a substance that inhibits translation in which CDKAL1 is involved. That is, the present invention is, in another aspect, a method for screening a substance that inhibits translation in which CDKAL1 is involved.
  • the step of introducing the nucleic acid construct encoding the RNA construct having, into a cell (2) A step of contacting the cells into which the nucleic acid construct has been introduced with a solution containing a test substance or a solution not containing the test substance. (3) A step of measuring the intensity of a signal derived from the reporter protein in the cells contacted with the solution containing the test substance, and (4) A step of comparing the measured intensity of the signal with the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing no test substance.
  • the present invention relates to a screening method comprising.
  • the type of substance to be screened there is no particular limitation on the type of substance to be screened, and it may be a natural substance or a synthetic substance, and an organic compound or an inorganic compound. May be there.
  • the molecular weight of the substance to be screened is not particularly limited, and may be a low molecular weight compound having a molecular weight of about 500, a medium molecular weight compound having a molecular weight of about 500 to 1000, or a high molecular weight compound having a molecular weight higher than that.
  • the substance to be screened may be provided as a library such as a small molecule compound library, a medium molecule compound library, a peptide library, and an antibody library. If the efficiency of introducing these substances into cells is low, screening may be performed using an appropriate delivery means such as liposomes.
  • Step (1) Step of introducing a nucleic acid construct into a cell This is a step of introducing the nucleic acid construct into a cell by bringing the nucleic acid construct into contact with the cell.
  • the RNA construct is produced intracellularly.
  • the RNA construct produced in the cell is translated by a translation mechanism involving CDKAL1, a reporter protein encoded by the first RNA sequence is produced.
  • the nucleic acid construct can be introduced into cells by an appropriate method regardless of a physical method or a chemical method, depending on the type of cell to which the nucleic acid construct is introduced and the environment in which the cells are present.
  • the appropriate method may be selected.
  • Examples of the method for introducing a physical nucleic acid construct include an electroporation method, a sonoporation method, and a microinjection method.
  • As a method for introducing a chemical nucleic acid construct in addition to the calcium phosphate method and the lipofection method using liposomes, cationic lipids, lipidoids, cationic polymers, membrane-permeable peptides, antibodies, antibody fragments, proteins, and nanoparticles. , Microparticles, emulsions and the like, the transfection method using an appropriate delivery means is exemplified.
  • the cells into which the nucleic acid construct has been introduced are preferably used in the step (2) described later after the incubation for a predetermined time after the step (1). That is, from the viewpoint of sufficiently introducing the nucleic acid construct into the cell and sufficiently producing the RNA construct in the cell, it is preferable to incubate the nucleic acid construct for 6 hours or more after contacting the cell. It is preferable to incubate for 12 hours or longer, more preferably 24 hours or longer, and even more preferably 48 hours or longer.
  • Step (2) Step of contacting cells with a solution containing a test substance or a solution not containing a test substance
  • a solution containing a test substance to be screened or a solution containing the test substance to be screened on the cells contacted with the nucleic acid construct As a control, it is a step of contacting a solution containing no test substance. That is, in the cell into which the nucleic acid construct was introduced in the step (1), the cell is brought into contact with the test substance in a state where the RNA construct is present in the cell.
  • the solution containing the test substance may be prepared by dissolving the test substance to be screened in water, a buffer solution, a physiological saline solution, or an appropriate solvent such as a cell culture medium.
  • the test substance to be screened is insoluble in water, it may be prepared by dissolving it in an organic solvent having low cytotoxicity such as DMSO and being miscible with water.
  • the solution containing the test substance can be brought into contact with the cells.
  • a cell culture medium can be used, but it is preferable to use the same solvent as the solvent used to prepare the solution containing the test substance.
  • the cells can be brought into contact with the solution containing no test substance by adding a predetermined amount of the solution containing no test substance to the cell culture solution.
  • the amount of the solution containing the test substance added is equal to the amount of the solution containing the test substance.
  • step (3) incubation for a predetermined time after the step (2). After that, it is preferable to use it in the step (3) described later. For example, it is preferable to use it in step (3) after incubating for 6 hours or more after contacting the cells with the solution containing or not containing the test substance, and more preferably after incubating for 12 hours or more, and incubating for 18 hours or more. It is more preferable to use it after incubating for 24 hours or more, and it is further preferable to use it after incubating for 24 hours or more.
  • Step (3) Measuring the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing the test substance
  • the cells contacted with the solution containing the test substance to be screened This is a step of quantitatively evaluating the intensity of a signal derived from a reporter protein that reflects the expression level of the reporter protein encoded by the RNA construct.
  • the RNA sequence represented by the above formula 1 and / or the above formula 2 is represented on the upstream side (5'end side) of the first RNA sequence encoding the reporter protein and on the 5'end side thereof.
  • the RNA construct having a second RNA sequence containing the RNA sequence is translated by a translation mechanism involving CDKAL1. Therefore, the expression level of the reporter protein encoded by the RNA construct, i.e., the intensity of the signal derived from the reporter protein, reflects the activity of the translation mechanism involved in CDKAL1 in the cell.
  • the intensity of the signal derived from the reporter protein can be measured by an appropriate means according to the type of the reporter protein used in the screening method.
  • a luminescent enzyme protein such as firefly luciferase, sea urchin luciferase, or marine luciferase
  • a substrate having the property of emitting light by receiving an enzymatic reaction by the luminescent enzyme protein for example, luciferin
  • the intensity of the signal derived from the reporter protein can be measured.
  • a fluorescent protein such as blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)
  • BFP blue fluorescent protein
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • RFP red fluorescent protein
  • the fluorescence intensity emitted by the fluorescent protein is emitted. May be measured using a fluorescent photometer.
  • a color-developing enzyme protein such as ⁇ -galactosidase is used as a reporter protein
  • a substrate having the property of absorbing light of a specific wavelength or emitting light by receiving an enzymatic reaction by the color-developing enzyme protein for example, for example.
  • the chromogenic enzyme protein is ⁇ -galactosidase, 5-bromo-4-chloro-3-indrill- ⁇ -D-galactopyranoside, 2-nitrophenyl- ⁇ -D-galactopyranoside, fluorescein- By using ⁇ -D-galactopyranoside, etc.) and an absorptiometer for measuring the absorbance, or a fluorescence photometer for measuring the fluorescence intensity, the intensity of the signal derived from the reporter protein can be measured.
  • the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing the test substance is also the same as the measurement of the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing the test substance. Needless to say, it is measured.
  • Step of comparing the measured intensity of the signal with the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing no test substance the nucleic acid construct or the above.
  • the expression level of the reporter protein encoded by the RNA construct i.e., the intensity of the signal derived from the reporter protein, reflects the activity of the translation mechanism involved in CDKAL1 in the cell. Therefore, by comparing the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing the test substance with the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing no test substance. , The effect of the test substance to be screened on the activity of the translation mechanism involved in CDKAL1 can be evaluated.
  • the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing the test substance is the signal derived from the reporter protein in the cells contacted with the solution containing the test substance.
  • the test substance is a substance having an action of suppressing the translation mechanism in which CDKAL1 is involved.
  • test substance in which the intensity of the signal is reduced by 10% or more as compared with the intensity of the signal derived from the reporter protein in the cells contacted with the solution containing no test substance, and more preferably 20%. As described above, it is more preferable to select a test substance having a reduction of 30% or more.
  • a pLKO.1 puro-shRNA plasmid expressing shRNA targeting each of CDKAL1, eIF4E, and eIF4G was prepared according to a conventional method.
  • SEQ ID NO: 1 and FIG. 27A of the sequence listing and the two types of DNA sequences shown in SEQ ID NO: 5 and FIG. 27B of the sequence listing were used.
  • the base sequence underlined in FIGS. 27A and 27B is a region encoding an RNA sequence complementary to the mRNA of CDKAL1.
  • shRNA encoded by the DNA sequence of SEQ ID NO: 1 in the sequence listing is referred to as “shCDKAL1 # 1”
  • shRNA encoded by the DNA sequence of SEQ ID NO: 5 in the sequence listing is referred to as “shCDKAL1 # 2”.
  • the RNA sequences of shCDKAL1 # 1 and shCDKAL1 # 2 are shown in FIG. 27A and SEQ ID NO: 2, and FIG. 27B and SEQ ID NO: 5, respectively.
  • 293FT cells (Thermo Fisher Scientific, Catalog No .: R70027) were cultured on a cell culture dish having a diameter of 10 cm so as to have a cell density of 80%.
  • fetal bovine serum (Corning, catalog number: 35-079-CV) with a final concentration of 10%
  • penicillin-streptomycin-L-glutamine solution (x100) (Fuji Film Wako Pure Chemical Industries, Ltd., catalog number) : 161-23201) was added so as to have a final concentration ⁇ 1 (high glucose) (containing L-glutamine and phenol red) (Fuji Film Wako Junyaku Co., Ltd., Catalog No .: 048-29763) (hereinafter , This mixed medium is sometimes called "basic medium").
  • the plasmid prepared in the above experiment was transfected into 293FT cells. That is, pLKO.1 puro-shRNA plasmid (10 ⁇ g) expressing shRNA targeting CDKAL1, eIF4E or eIF4G and psPAX2 (psPAX2) were added to 293FT cells cultured on a cell culture dish having a diameter of 10 cm so as to have a cell density of 80%.
  • Addgene, catalog number: 12260) (7.5 ⁇ g)
  • pMD2.G addgene, catalog number: 12259) (2.5 ⁇ g) were transfected by lipofection method, followed by 37 ° C., 5% CO.
  • the lentiviral vectors expressing shCDKAL1 # 1 or shCDKAL1 # 2 obtained by the above procedure are referred to as Lenti-shCDKAL1 # 1 and Lenti-shCDKAL1 # 2, respectively.
  • a outside virus vector expressing shRNA having a scrambled sequence that does not target any gene hereinafter referred to as “shControl”. This lenti-virus vector is called Lenti-shControl.
  • Glioma stem cells corresponding to "JKGIC2". Further, in the following procedure, the malignant brain tumor cell line JK2 has B-27 Supplement (50x), Serum Free (Thermo Fisher Scientific, Catalog No .: 17504044) at the final concentration ⁇ 1, N-2 Supplement ( ⁇ 100) (ThermoFis).
  • EGF Epithelial cell growth factor
  • Neurobasal medium ThermoFisher Scientific, Catalog No .: 21103049
  • this mixed medium may also be referred to as "JK2 basic medium”
  • JK2 basic medium added to a concentration of 002 mg / mL.
  • JK2 cells were infected with a lentiviral vector by the procedure shown below. That is, a lentiviral vector or any gene expressing shRNA targeting CDKAL1, eIF4E, or eIF4G prepared in Experiment 1 in a suspension of about 500,000 JK2 cells in 4 mL of JK2 basal medium. 1 mL of a lentiviral vector-containing medium containing a lenti-sh Control not targeted was added to make a total of 5 mL. The mixed solution was seeded in a cell culture dish having a diameter of 60 mm and incubated in an environment of 37 ° C. and 5% CO 2 for 4 days according to a conventional method.
  • RNaseA Sigma-Aldrich, Catalog No .: R6513
  • concentration in the cell lysate 100 ⁇ g / mL.
  • RNaseA was added for the purpose of removing RNA and promoting the formation of a translation initiation factor complex.
  • the obtained cytolytic solution (containing 1.5 mg of protein) was washed with an immunoprecipitation buffer, and the immunoprecipitation buffer was adjusted to twice the initial amount.
  • 60 ⁇ L of m7GTP beads product name “Immobilated ⁇ -Aminophenyl-m7GTP (C10-spacer)”, Jena Bioscience, Catalog No .: AC-155S) resuspended in 1 was added and mixed at 4 ° C. for 16 hours.
  • CDKAL1 antibodies product name "CDKAL1 antibody rabbit polyclonal", Proteintech, Catalog No .: 22988-1-, respectively.
  • eIF4E antibody product name "eIF4E (C46H6) Rabbit mAb", Cell Signaling technology, catalog number: 2067
  • eIF4A antibody product name "eIF4A (C32B4) Rabbit mAb", Cell signal 2013
  • eIF4G antibody product name "eIF4G (C45A4) Rabbit mAb", Cell Signaling technology, catalog number: 2469
  • the secondary antibody is an HRP-labeled anti-rabbit IgG antibody (product name "Anti-rabbit IgG”).
  • HRP-linked Antibody was used. Detection was performed by using Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Catalog No .: 1705062) and ChemiDoc Touch Imaging System (Bio-Rad Laboratories) according to the respective instructions.
  • FIG. 1 The results of Western blotting are shown in Fig. 1.
  • a cytolytic solution of JK2 cells infected with Lenti-shCDKAL1 # 1 (a cytolytic solution before protein recovery by m7GTP beads.
  • Lenti-shCDKAL1 # 1 a cytolytic solution before protein recovery by m7GTP beads.
  • FIG. 1 corresponds to "Input” and shRNA "CDKAL1”.
  • the intensity of the band corresponding to CDKAL1 was significantly diminished as compared to the cytolytic solution obtained from JK2 cells infected with Lenti-shControl. This result indicates that infection with Lenti-shCDKAL1 # 1 reduces the expression level of CDKAL1 in the cells, that is, the expression of CDKAL1 is knocked down.
  • CDKAL1 is essential for the formation of a translation initiation factor complex formed by binding eIF4E to eIF4A and eIF4G in cancer stem cells. It is not known at all that CDKAL1 is involved in the formation of the translation initiation factor complex, and the above results are completely surprising findings for the present inventors.
  • the resulting cell suspension was placed in a 24-well ultra-low adhesive plate (product name "Costar ultra-low adhesive surface plate with 24-well flat bottom lid", Corning, Catalog No .: 3473) in an amount of 2 mL per well.
  • a total of 4 wells were sown and incubated for 1 week in an environment of 37 ° C. and 5% CO 2 according to a conventional method. After 1 week of incubation, the number of spheres formed was visually counted. After measuring the number of spheres, cells were isolated by allowing trypsin to act on the formed spheres. The isolated cells were suspended again in JK2 basal medium, seeded and incubated for 1 week according to the procedure described above, and then the number of spheres formed was visually counted. The results are shown in FIG.
  • a chamber slide coated with a Matrigel basement membrane matrix (Corning, Catalog No .: 356231, diluted with PBS to a final concentration of 10% and used for coding) (product name "Lab-TekII Chamber Slide System”).
  • Thermo Fisher Scientific, Catalog No .: 154534 PK suspended JK2 cells were seeded to a cell count of about 50,000. After incubating for 24 hours in an environment of 37 ° C. and 5% CO 2 , the cell culture medium was removed, washed once with PBS, and then 4% paraformaldehyde / phosphate buffer (Fuji Film Wako Junyaku Co., Ltd.).
  • the primary antibody used in the above procedure was a rabbit anti-Vimentin antibody (product name "Vimentin (D21H3) XP Rabbit mAb", Cell Signaling Technology, catalog number: 5741), anti-MSI1 antibody (product name "Human”). / Mouse / Rat Musashi-1 Antibody ”, R & D Systems, Catalog number: AF2628), Goat anti-SOX2 antibody (Santa Cruz Biotechnology, Catalog number: sc-17320), Rabbit anti-Nestin antibody (product name: anti-Nestin antibody).
  • Rabbit host antibody ”, Sigma-Aldrich, catalog number: N5413
  • mouse anti-SYS antibody product name“ monoclonal anti-synaptophidin mouse host antibody ”, Sigma-Aldrich, catalog number: S5768
  • anti-MAP2 antibody Santa
  • Cruz Biotechnology Catalog No .: sc-5359
  • PBS containing antibody diluted solution 3% bovine serum albumin and 0.01% Triton X-100
  • the cells were washed 3 times with PBS and the secondary antibody diluted with the antibody diluent was reacted in a moist environment at room temperature for 1 hour.
  • the secondary antibody used was an anti-rabbit, anti-mouse, or anti-goat antibody labeled with Alexa488 or Alexa594, all of which were diluted with an antibody diluent so that the final antibody concentration was 10 ⁇ g / mL.
  • the encapsulation was performed by using according to the document. After allowing to stand at room temperature for 24 hours to fix the encapsulant, an image was acquired using a confocal laser scanning microscope FV3000 (manufactured by Olympus Co., Ltd.). Shown in 4.
  • the RD infected with the lentiviral vector was used in the same manner as in Experiment 3 except that the horizontal print myoma cell line RD (purchased from JCRB cell bank, cell number: JCRB9072) was used instead of JK2 cells.
  • the ability of cells to form spheres was evaluated. That is, a medium containing a lentivirus vector containing either Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl in a medium in which about 500,000 RD cells are suspended in 4 mL of basal medium. 1 mL was added to make a total of 5 mL, and the mixture was seeded in a 60 mm cell culture dish.
  • the sphere-forming ability of RD cells infected with the lentiviral vector was evaluated according to the same procedure as described in Experiment 3. The obtained results are shown in FIG. 5A.
  • CDKAL1 is an essential factor for maintaining the self-renewal ability of many cancer stem cells, including not only malignant brain tumor cells JK2 but also rhabdomyosarcoma cells RD cells. Is shown.
  • Suspended cells were seeded in 6-well cell culture plates for a total of 3 wells so that the number of cells per well was about 2,000.
  • the amount of basal medium per well is 3 mL.
  • Two weeks after sowing, fixation and staining were performed according to a conventional method, and the number of colonies remaining on the surface of the cell culture plate was visually measured. The obtained results are shown in FIG. 5B.
  • the sources of the cells used in this experiment are as follows: Human malignant melanoma cells A2058 (obtained from JCRB cell bank, model number: IFO50276), SK-Mel-28 (obtained from ATCC, model number: HTB-).
  • HMV-II (Riken BioResource Research Center, model number: RCB0777), human liver cancer cell Huh-7 (obtained from JCRB cell bank, model number: JCRB0403), HepG2 (obtained from JCRB cell bank, model number: JCRB1054) ), Human prostate cancer cell PC3 (obtained from ATCC, model number: CRL-3470 TM ), LNCaP (Riken BioResource Research Center, model number: RCB2144), human gastric cancer cell NUGC3 (obtained from JCRB cell bank, model number: JCRB0822), HGC27 (obtained from Riken BioResearch Research Center, model number: RCB20500), MKN45 (obtained from JCRB cell bank, model number: JCRB0254).
  • the expression of cancer stem cell markers after knocking down CDKAL1 was observed using a confocal microscope.
  • the procedure is the same as the procedure described in Experiment 5 except that the above cells were used instead of JK2 cells and the antibody used for immunostaining was appropriately changed.
  • the primary antibodies used were as follows: anti-ALDH1A1 antibody (Novus Biologicals, catalog number: NBP1-89152), anti-CD44 antibody (BioLegend, catalog number: 103001), anti-CD133 antibody (Proteintech, Inc., Catalog number: 66666-1-Ig).
  • human malignant brain tumor cells MGG4, MGG8, and MGG18 the expression level of cancer stem cell markers after knockdown of CDKAL1 was evaluated by Western blotting.
  • human malignant brain tumor cells MGG4, MGG8, and MGG18 were infected with a lentiviral vector expressing shCDKAL1 according to the procedure of Experiment 6. That is, a medium containing a lentivirus vector containing Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl as a control in a medium in which about 500,000 cells are suspended in 4 mL of the medium having the above composition.
  • the antibodies used for detection were GAPDH antibody (ProteinTech, catalog number: 6004-1-Ig), SOX2 antibody (Santa Cruz Biotechnology, catalog number: sc-17320), CD133 antibody (ProteinTech, catalog number:). 18470-1-AP), POU3F3 antibody (Cell Signaling Technology, catalog number: 12137S), OLIG2 antibody (Abcam, catalog number: ab109186), HRP-labeled anti-rabbit IgG antibody (Cell Signaling Technology, catalog number: 74).
  • HRP-labeled anti-mouse IgG antibody (Sigma Aldrich, catalog number: A9044), HRP-labeled anti-goat IgG antibody (Sigma Aldrich, catalog number: A4174), and HRP-labeled anti-rat IgG antibody (Sigma Aldrich, catalog number). : A5795). Detection was performed by using Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Catalog No .: 1705062) and ChemiDoc Touch Imaging System (Bio-Rad Laboratories) according to the respective instructions.
  • FIGS. 6 to 10 The obtained results are shown in FIGS. 6 to 10.
  • malignant melanoma FIGS. 6 to 10
  • liver cancer FIG. 7
  • prostate cancer FIG. 8
  • gastric cancer FIG. 9
  • malignant brain tumor FIG. 10
  • the sphere-forming ability which is an index of self-renewal ability
  • the colony-forming ability which is an index of tumor-forming ability
  • attenuation of the expression of cancer stem cell markers was observed in all cancer types. It has been shown that in all cancer types, antitumor activity can be obtained by inhibiting the translation mechanism in which CDKAL1 is involved.
  • RMS-YM cells were harvested using trypsin according to conventional methods, and the recovered cells were resuspended in PBS to a cell concentration of 1 million cells / 100 ⁇ L. 100 ⁇ L of the obtained suspension (corresponding to 1 million cells) is injected subcutaneously into a 5-week-old female BALB / c-nu / nu mouse, followed by the time course of tumor size at the injection site. Changes were evaluated according to the report of Wu et al. (Wu W. et al., Clinical Cancer Research 2013 Oct 15; 19 (20): 5699-5710).
  • the maximum diameter of the tumor (Length) and the tumor diameter (Width) in the direction perpendicular to the maximum diameter are measured with a nogis over time, and the measurement results are applied to the following formula to measure the volume of the tumor (Tumor Volume). ) was asked.
  • Pi represents pi
  • Length represents the maximum diameter of the tumor
  • Width represents the tumor diameter in the direction perpendicular to Length.
  • Figure 11 shows the change in tumor size over time.
  • FIG. 11 in mice injected with RMS-YM cells infected with a lentivirus vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2) and knocked down CDKAL1, Lenti-shControl Significant suppression of tumor growth was observed compared to mice injected with RMS-YM cells infected with.
  • This result indicates that knockdown of CDKAL1 significantly reduces the growth ability of tumors formed from RMS-YM cells, which are rhabdomyosarcoma cells, and knockdown of CDKAL1 significantly reduces the ability to grow tumors at the in vivo level. Also show that a remarkable antitumor effect can be obtained.
  • cytolytic fluid for obtaining a polysome fraction from RD cells was collected according to the following procedure.
  • cycloheximide was added to the cell culture medium on the cell culture dish at a rate of 100 ⁇ g / mL and incubated at 37 ° C. for 5 minutes.
  • the cells were then lysed with RNA Lysis Buffer (15 mM Tris-HCl (pH 7.4), 15 mM MgCl 2 , 0.3 M NaCl, 1% TritonX-100, 0.1 mg / mL cycloheximide, 100 units / mL RNase inhibitor).
  • the obtained cell lysate was fractionated by sucrose density gradient (10-50%) ultracentrifugation. Ultracentrifugation was performed at 39,000 rpm for 90 minutes at 4 ° C.
  • RNA recovered from the heaviest fraction (9th to 11th fractions) is analyzed by RNA sequence as the most actively translated RNA (Actively Translated RNA), and as a comparison, cell lysis before fractionation is performed.
  • Total RNA contained in the fluid was analyzed by RNA sequence. The acquisition of RNA sequence information was outsourced to Rhelixa Co., Ltd.
  • the evaluation of the obtained analysis results is performed by plotting the amount of change in the expression level of RNA in cells on the horizontal axis and the amount of change in the amount of RNA contained in the polysome fraction on the vertical axis for RNA of each gene. gone.
  • the amount of change in the expression level of RNA in the cell is the total amount recovered from the cell lysate of RD cells infected with the lentivirus vector expressing shCDKAL1 without undergoing a fractionation step.
  • the fact that the expression level of RNA in a cell is changed by knockdown of CDKAL1 indicates that the gene corresponding to the RNA is regulated at the transcription level.
  • the amount of change in the amount of RNA contained in the polysome fraction is the RNA contained in the polysome fraction recovered from the cell lysate of RD cells infected with a lentivirus vector expressing shCDKAL1.
  • shCDKAL1 the fact that the amount of the RNA contained in the polysome fraction is changed even though the expression level of the RNA in the cell is not changed due to the knockdown of CDKAL1 corresponds to the RNA. It is shown that the gene is regulated at the translation level.
  • FIG. 12 shows the results of plotting the obtained measured values as described above.
  • infection with a lentiviral vector expressing shCDKAL1, that is, knockdown of CDKAL1 causes no change in the amount of RNA at the transcription level (the value on the horizontal axis does not change).
  • a plurality of gene groups in which the amount of RNA contained in the polysome fraction fluctuates (the value on the vertical axis fluctuates), that is, a group of genes subject to translation control by CDKAL1 have been identified.
  • FIG. 13 Among the gene clusters whose translation is suppressed by knockdown of CDKAL1 shown in FIG. 12 (that is, in FIG. 12, the gene cluster in which the value on the vertical axis decreases even though the value on the horizontal axis does not change). , Transcription factors were focused on and extracted, and changes in gene expression of each gene are shown in FIG. 13 as a heat map. As shown in FIG. 13, including SALL2, SP9, IRF2BPL, ZNF276, IFI35, YAP1, MIER1, HOXA7, PHF3, LBX2, KLF7, HOXB6, PLAG1, ZNF484, ZNF516, HLTF, HIC1, MAML2.
  • the translation inhibition rate was even higher for the SALL2, SP9, IRF2BPL, ZNF276, IFI35, YAP, and MIER1 genes.
  • Particularly strong translational repression was confirmed for the SALL2, SP9, IRF2BPL, ZNF276, and IFI35 genes, and the largest translational repression was observed for the SALL2 gene.
  • RD cells were infected with a lentiviral vector expressing shCDKAL1 and CDKAL1 was knocked down. That is, a medium containing Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl containing Lenti-shControl as a control was added to a medium in which about 500,000 RD cells were suspended in 4 mL of basal medium. 1 mL was added to make a total of 5 mL, and the mixture was seeded in a 60 mm cell culture dish.
  • cytolysates of RD cells were obtained and subjected to Western blotting according to the procedure described in Experiment 2.
  • the antibodies used for detection were GAPDH antibody (mouse monoclonal) (proteintech, catalog number: 6004-1-Ig), SALL2 antibody (Bethyl Laboratories, catalog number: A303-208A), HRP-labeled anti-rabbit IgG antibody. (Cell Signaling Technology, Catalog No .: 7074) and HRP-labeled anti-mouse IgG antibody (Cell Signaling Technology, Catalog No .: 7076).
  • FIG. 14A in RD cells infected with a lentiviral vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2), compared with RD cells infected with Lenti-shControl. It was confirmed that the expression of SALL2 was decreased. This result indicates that knockdown of CDKAL1 reduces the expression level of SALL2 at the protein level.
  • FIG. 14B when the expression level of the mRNA corresponding to SALL2 was confirmed by RT-PCR in the RD cells in which CDKAL1 was knocked down, the decrease in the expression of the SALL2 gene was observed at the level of the mRNA expression level. Not confirmed. That is, it was suggested that the decrease in the expression level of SALL2 confirmed by Western blotting was due to the suppression of translation by knockdown of CDKAL1.
  • shRNA encoded by the nucleotide sequence of SEQ ID NO: 9 and FIG. 28A in the sequence listing is referred to as “shSALL2 # 1”
  • shRNA encoded by the nucleotide sequence of SEQ ID NO: 13 and FIG. 28B of the sequence listing is referred to as “shSALL2 #”. 2 ".
  • the RNA sequences of shSALL2 # 1 and shSALL2 # 2 are as shown in FIGS. 28A and 10 and 28B and 14 respectively.
  • the lentiviral vectors expressing shSALL2 # 1 or shSALL2 # 2 are referred to as Lenti-shSALL2 # 1 and Lenti-shSALL2 # 2, respectively.
  • the RD cells were infected with the lentivirus vector expressing shSALL2 according to the procedure described in Experiment 6, except that the lentiviral vector expressing shSALL2 was used instead of the lentiviral vector expressing shCDKAL1. ..
  • Four days after infection RD cells infected with a lentiviral vector expressing shSALL2 were evaluated for sphere-forming ability and colonization ability of the RD cells according to the procedure described in Experiment 6. The obtained results are shown in FIGS. 14C and 14D.
  • Lenti-shControl was used in mice infected with a lentiviral vector expressing shSALL2 (Lenti-shSALL2 # 1 or Lenti-shSALL2 # 2) and injected with RMS-YM cells in which SALL2 was knocked down.
  • Significant suppression of tumor growth was observed as compared to mice injected with RMS-YM cells infected with.
  • This result indicates that knockdown of SALL2 significantly reduces the growth ability of tumors formed from RMS-YM cells, which are rhabdomyosarcoma cells, and knockdown of SALL2 significantly reduces the ability to grow tumors at the in vivo level. Also show that a remarkable antitumor effect can be obtained.
  • a plasmid (pTomo-HRas / hp53) constructed to express HRas in response to the expression of Cre recombinase and to express shRNA against the cancer suppressor gene p53 according to a conventional method (vector skeleton: obtained from pTomo vector (Addgene), Catalog number: 26291)) (FIG. 16A) was prepared, a lentiviral vector incorporating the plasmid was prepared according to the procedure of Experiment 1, and the obtained lentiviral vector was infected with C2C12 cells.
  • an adenovirus expressing Cre recombinase (Vector Biolabs, catalog number: 1045) was further infected at a ratio of 10 MOI (multiplicity of infection). After infecting with the adenovirus, the cells were cultured for another 7 days to obtain HRas / skip53-C2C12.
  • Control-C2C12 into which a control vector (pTomo-RFP-ires-GFP) was introduced into C2C12 cells instead of pTomo-HRas / shp53 was obtained according to the above procedure.
  • the medium used for cell culture in the above procedure is a basal medium.
  • Control-C2C12 cells did not form a tumor mass when subcutaneously infused into immunocompromised mice, whereas HRas / shp53-C2C12 cells were histopathologically rhabdomyosarcoma. Formed a tumor mass very similar to. This confirmed the establishment of a malignant tumor cell model and a normal cell model.
  • the expression level of CDKAL1 was increased in HRas / shop53-C2C12 cells as compared with Control-C2C12 cells. As a result, it was found that the expression level of CDKAL1 was increased in C2C12 cells in the process of malignant transformation.
  • the sphere forming ability was evaluated as an index of the self-renewal ability of Control-C2C12 cells and HRas / skip53-C2C12 cells. Specifically, Control-C2C12 cells and HRas / skip53-C2C12 cells were used instead of JK2 cells, and the sphere forming ability was determined in the same manner as in the procedure of Experiment 3 except that the lentiviral vector was not infected. evaluated. In this experiment, in addition to measuring the number of spheres, the state of sphere formation was photographed using an inverted research microscope (model number "IX71", Olympus Corporation). The obtained results are shown in FIG. 16D.
  • Control-C2C12 cells do not form spheres, i.e., do not have self-renewal ability, whereas HRas / skip53-C2C12 cells form spheres, i.e. acquire self-renewal ability. It became clear that they were doing it.
  • a lentiviral vector for expressing shRNA specific to mouse CDKAL1 was prepared according to the procedure described in Experiment 1. That is, as the DNA sequence encoding the shRNA targeting CDKAL1, instead of the DNA sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 5, SEQ ID NO: 17 or SEQ ID NO: 18, which is a base sequence specific to mouse CDKAL1, is used. A lentivirus vector was prepared in the same procedure as shown in Experiment 1 except that the described DNA sequence was used.
  • the lenti-mouse shCDKAL1 # 1 lenti-mouse shCDKAL1 prepared using the DNA sequence set forth in SEQ ID NO: 17 and the lenti-mouse shCDKAL1 prepared using the DNA sequence set forth in SEQ ID NO: 17 are used. # 2 ".
  • the knockdown of CDKAL1 and the evaluation of the sphere forming ability were performed according to the procedure described in Experiment 3. That is, except that Control-C2C12 cells and HRas / shp53-C2C12 cells were used instead of JK2 cells, and Lenti-mouse shCDKAL1 # 1 or Lenti-mouse shCDKAL1 # 2 was used as the lenti-mouse shCDKAL1 # 2, in Experiment 3.
  • the cells were infected with a lentiviral vector, knocked down CDKAL1, and then evaluated for sphere-forming ability in the same procedure as described. Further, after infecting the lentiviral vector in the same manner and knocking down CDKAL1, the expression level of CDKAL1 was evaluated by Western blotting according to the procedure shown in Experiment 2. The obtained results are shown in FIG. 16E.
  • the knockdown of CDKAL1 and the evaluation of the sphere forming ability were performed according to the procedure described in Experiment 6. That is, Experiment 6 except that Control-C2C12 cells and HRas / shp53-C2C12 cells were used instead of RD cells, and Lenti-mouse shCDKAL1 # 1 or Lenti-mouse shCDKAL1 # 2 was used as the lenti-mouse shCDKAL1 # 2 as a lenti-virus vector. Control-C2C12 cells and HRas / skip53-C2C12 cells were infected with a lentiviral vector in the same procedure as described in 1 and evaluated for colony viability. The obtained results are shown in FIG. 16F.
  • CDKAL1 is not essential for the maintenance and proliferation of normal cells, whereas CDKAL1 is essential for the maintenance and proliferation of cancerous cells. That is, suppression of CDKAL1 expression, inhibition of translation involving CDKAL1, or suppression of expression of a gene translated with CDKAL1 involved has a very small effect on normal cells, and is a low-toxic and effective cancer. It has been shown that it can be a means of treatment.
  • CDKAL1 has an UPF domain, a Radical SAM domain, a TRAM domain, and a Hydrophobic domain in order from the amino-terminal (N-terminal) side. ..
  • various CDKAL1 variants in which each domain was deleted or mutated were prepared, and which domain of CDKAL1 was important for the stabilizing action of the translation initiation factor complex by CDKAL1 was examined.
  • CDKAL1 WT represents wild-type CDKAL1 and has the amino acid sequence represented by SEQ ID NO: 19.
  • CDKAL1 6CA is a variant in which 6 cysteines in the UPF domain and Radical SAM domain are replaced with alanine, and lacks the function as a tRNA modifying enzyme.
  • the amino acid sequence is as set forth in SEQ ID NO: 21.
  • CDKAL1 deltaC , CDKAL1 deltaTC , CDKAL1 UPF , and CDKAL1 deltaN are deletion variants as shown in FIG. 17B.
  • CDKAL1 UPF deletes the amino acid sequence on the C-terminal side including the Radical SAM domain, TRAM domain and Hydrophobic domain
  • CDKAL1 deltaN deletes the amino acid sequence on the N-terminal side including the UPF domain.
  • the amino acid sequences of CDKAL1 deltaC , CDKAL1 deltaTC , CDKAL1 UPF , and CDKAL1 deltaN are as set forth in SEQ ID NOs: 23, 25, 27, and 29, respectively.
  • Lentiviral vectors expressing each variant of CDKAL1 WT and CDKAL1 were prepared according to the procedure described in Experiment 1. That is, pLKO. Instead of 1 puro-SHRNA plasmid (10 ⁇ g), pTomo plasmid (10 ⁇ g; vector skeleton (pTomo vector) was obtained from Addgene, catalog number: 26291) in which the DNA sequence encoding each CDKAL1 mutant was inserted was obtained from psPAX2 (7. 5 ⁇ g) and pMD 2. It was prepared in the same manner as in Experiment 1 except that it was transfected into 293FT cells together with G (2.5 ⁇ g).
  • CDKAL1 WT The DNA sequences encoding CDKAL1 WT , CDKAL1 6CA , CDKAL1 deltaC , CDKAL1 deltaTC , CDKAL1 UPF , and CDKAL1 deltaN are as described in SEQ ID NOs: 20, 22, 24, 26, 28, and 30, respectively. be.
  • CDKAL1 variant on translation initiation factor complex stabilizing action CDKAL1 expression in RD cells in which the above lentivirus vector was infected with RD cells and overexpressed wild-type CDKAL1 or CDKAL1 variant.
  • the variant of CDKAL1 can stabilize the translation initiation factor complex. We verified whether it would contribute. The specific procedure is as shown below.
  • RD cells were infected with a lentiviral vector to express wild-type CDKAL1 or CDKAL1 mutants. That is, wild-type CDKAL1, a variant of CDKAL1, or green fluorescent protein as a negative control in a cell suspension in which approximately 500,000 (5 ⁇ 10 5 ) RD cells were suspended in 4 mL of basal medium. 1 mL of a lentiviral vector-containing medium containing a lentiviral vector expressing (GFP) was added to make a total of 5 mL. The mixed solution was seeded in a cell culture dish having a diameter of 60 mm and incubated in an environment of 37 ° C. and 5% CO 2 for 3 days according to a conventional method. This overexpressed wild-type CDKAL1, a mutant of CDKAL1, or GFP in RD cells.
  • wild-type CDKAL1, a variant of CDKAL1, or green fluorescent protein as a negative control in a cell suspension in which approximately 500,000 (5 ⁇ 10 5 )
  • a lenti-shCDKAL1 # 1 lentiviral vector (Lenti-shCDKAL1 # 1) expressing shRNA targeting CDKAL1 prepared in Experiment 1 was applied to RD cells expressing wild-type CDKAL1, a mutant of CDKAL1, or GFP. Infected. That is, three days after infection with the wild-type CDKAL1, a mutant of CDKAL1, or a lentiviral vector expressing GFP, Lenti-shCDKAL1 # 1 or Lenti-shControl as a negative control was further infected.
  • a lentiviral vector-containing medium containing Lenti-shCDKAL1 # 1 or Lenti-shControl was added to a cell suspension in which approximately 500,000 RD cells were suspended in 4 mL of basal medium, for a total of. The volume was 5 mL.
  • the mixed solution was seeded in a cell culture dish having a diameter of 60 mm and incubated in an environment of 37 ° C. and 5% CO 2 for 3 days according to a conventional method. After incubation for 3 days, precipitation with m7GTP beads and Western blotting were performed in the same procedure as shown in Experiment 2. The obtained results are shown in FIG. 17C.
  • a mutant library in which amino acids that may undergo post-translational modification shown in FIG. 18A were mutated was prepared.
  • Variants were prepared by site-directed mutagenesis according to a conventional method. More specifically, for the amino acid to which the mutation is to be introduced, oligo primers (forward primer and reverse primer) designed to contain the post-mutation DNA sequence in the center were prepared, and PCR was performed using pTomo-CDKAL1 WT as a template. ..
  • the reagent used for PCR was KOD FX (Toyobo Co., Ltd., catalog number: KFX-101), and was used according to the package insert attached to the product.
  • coli-containing solution was seeded on an LB plate containing 50 ⁇ g / mL ampicillin and cultured at 37 ° C. for 16 hours. The next day, the colonies formed on the LB plate were collected, mixed with LB medium containing 50 ⁇ g / mL ampicillin, and further cultured at 37 ° C. for 16 hours. From the obtained culture medium, a DNA plasmid was purified according to a conventional method, and it was confirmed by DNA sequence analysis that the mutation was introduced.
  • the CDKAL1 mutant having the S18A / S22A, N107Q, and S153A mutations could not rescue the decrease in self-renewal ability due to the suppression of CDKAL1 expression by RNAi. rice field.
  • the above results indicate that the stabilizing effect of the translation initiation factor complex is impaired in the CDKAL1 mutant having the S18A / S22A, N107Q, and S153A mutations.
  • RD cells were seeded in a culture dish having a diameter of 35 mm so as to be about 80% confluent.
  • tunicamycin (CAS number: 11089-65-9) as an N-linked glycosylation inhibitor
  • BIO (6-bromoindirubin-30-oxime, CAS number: 667463-62-9) as an inhibitor of phosphorylation by GSK3.
  • CHIR-98014 (CAS number: 252935-94-7), or K252a (CAS number: 97161-97-2) as an inhibitor of phosphorylation by phosphorylase kinase was replaced with a medium containing a concentration of 5 ⁇ M, 37.
  • RNA and protein were extracted according to a conventional method and subjected to cDNA preparation and Western blotting for quantitative PCR, respectively.
  • RNA extraction was used according to the package insert attached to the product.
  • the extracted RNA was treated with DNase I (Takara Bio Inc., catalog number: 2270A, used according to the package insert), and cDNA was prepared based on this.
  • DNase I Takara Bio Inc., catalog number: 2270A, used according to the package insert
  • cDNA was prepared based on this.
  • PrimeScript RT Master Mix Takara Bio Inc., catalog number: RR036B was used according to the package insert attached to the product.
  • the obtained cDNA was subjected to quantitative PCR.
  • Quantitative PCR was performed using Luna Universal qPCR Master Mix (New England Biolabs, catalog number: M3003E) and Rotor-Gene Q (QIAGEN, catalog number: 9001630) according to the package insert attached to each product.
  • Western blotting was performed according to the procedure shown in Experiment 2. The obtained results are shown in FIG. 18C.
  • the specific experimental procedure is as follows.
  • the RD cells were cultured in the same procedure as described in Experiment 15.3, except that the RD cells were seeded in a 100 mm culture dish so as to be about 80% confluent instead of the 35 mm diameter culture dish. , Treated with each inhibitor. Then, according to the procedure described in Experiment 2, the protein was recovered, fractionated, and subjected to Western blotting. The obtained results are shown in FIG. 18D.
  • N-linked glycosylation inhibitors and GSK3 inhibitors showed that the above post-translational modifications were inhibited and that the expression of genes involved in the translation of CDKAL1 was effectively suppressed.
  • N-linked glycosylation inhibitors and GSK3 inhibitors having an action of inhibiting the translation mechanism involved in CDKAL1 can be suitably used for the treatment of rare cancers including rhabdomyosarcoma and malignant brain tumor.
  • the translation mechanism in which CDKAL1 is involved is considered to be an essential translation mechanism for the translation of specific genes such as SALL2.
  • the 5'untranslated region (5'UTR: 5'Untranslated Region) of the mRNA to be translated is cytosine (C) and guanine (5'UTR: 5'Untranslated Region). It is known to be rich in G) and have a characteristic secondary structure. Therefore, we have described 2 of the base sequence encoding the reporter protein and the base sequence of 5'UTR of mRNA translated by the translation mechanism involving CDKAL1 on the upstream side, that is, the 5'end side.
  • RNA construct having one base sequence it may be possible to easily evaluate the activity of the translation mechanism in which CDKAL1 is involved by measuring the production amount of the reporter protein which is the translation product of the RNA construct. It was verified by the experiment shown in.
  • RNA construct having a base sequence encoding firefly luciferase as a reporter protein and a base sequence of 5'UTR of the plasmid of SALL2 as an untranslated region on the upstream side, that is, on the 5'end side (upper part of FIG. 19).
  • a plasmid produced as a transcript in cells (hereinafter, this plasmid is referred to as "SALL2-5'UTR-firefly luciferase reporter plasmid”) was prepared according to a conventional method.
  • the base sequence encoding SALL2-5'UTR-firefly luciferase in the prepared SALL2-5'UTR-firefly luciferase reporter plasmid is as shown in SEQ ID NO: 31 in the sequence listing, and SALL2 is included in the base sequence.
  • the base sequence corresponding to the 5'untranslated region is as shown in SEQ ID NO: 32 of the sequence listing.
  • a plasmid that produces an RNA construct having a sequence (middle, bottom in FIG. 19) as a transcript in cells (hereinafter, these plasmids are referred to as "GAPDH-5'UTR-firefly luciferase reporter plasmid" or "ACTB-5'UTR-”.
  • a firefly luciferase reporter plasmid ) was prepared according to a conventional method.
  • the base sequence encoding GAPDH-5'UTR-firefly luciferase in the prepared GAPDH-5'UTR-firefly luciferase reporter plasmid is as shown in SEQ ID NO: 33 in the sequence listing.
  • the base sequence corresponding to the 5'untranslated region is as shown in SEQ ID NO: 34 in the sequence listing.
  • the base sequence encoding ACTB-5'UTR-firefly luciferase in the prepared ACTB-5'UTR-firefly luciferase reporter plasmid is as shown in SEQ ID NO: 35 in the sequence listing, and in the base sequence, ACTB The base sequence corresponding to the 5'untranslated region is as shown in SEQ ID NO: 36 of the sequence listing.
  • the lentiviral vector expressing shCDKAL1 was infected and the CDKAL1 was knocked.
  • the above plasmid was transfected into downed RD cells and RD cells infected with a lentiviral vector expressing shControl.
  • RD cells infected with a lentiviral vector expressing shCDKAL1 and incubated for 4 days were isolated by trypsin treatment according to a conventional method, and isolated RD cells.
  • 2 ⁇ g of SALL2-5'UTR-firefly luciferase reporter plasmid, GAPDH-5'UTR-firefly luciferase reporter plasmid, or ACTB-5'UTR-firefly luciferase reporter plasmid were efficiency of introducing the plasmid into cells.
  • pGL4.74 [hRluc / TK] vector Luciferase reporter plasmid As an index internal standard, 2 ⁇ g of pGL4.74 [hRluc / TK] vector Luciferase reporter plasmid (Promega, Catalog No .: E6921) was transfected. The transfection was performed using TransIT-LT1 Reagent (Takara Bio Inc., catalog number: MIR2300) as a lipofection reagent. The mixing ratio and procedure of each reagent followed the instructions attached to the product.
  • the luciferase activity in the cell into which each reporter plasmid was introduced is shown by the value obtained by dividing the luciferase activity of the sea urchin shiitake mushroom, which is an internal standard, by the luciferase activity.
  • RD cells were cultured in a 60 mm culture dish to a cell density of 80% confluent.
  • the medium used for cell culture is the basic medium.
  • 4 ⁇ g of SALL2-5'UTR-firefly luciferase reporter plasmid and 2 ⁇ g of pGL4.74 [hRluc / TK] vector Umi Shiitake mushroom luciferase reporter plasmid (Promega, Catalog No .: E6921) were transfected.
  • the transfection was performed using TransIT-LT1 Reagent (Takara Bio Inc., catalog number: MIR2300) as a lipofection reagent.
  • each reagent followed the instructions attached to the product.
  • luciferase activity was measured in the cells to which each test target compound was added, or to the cells to which the control substance DMSO was added as a control.
  • luciferase activity use the Dual-Glo Luciferase Assay System (Promega, catalog number: E2940) and luminometer (product name "MicroLumat Plus LB 96V", BERTHOLD) according to the respective package inserts. Was done by.
  • tunicamycin exerts a stronger inhibitory effect on translations involving CDKAL1 than Go6983. Therefore, the effect of tunicamycin, which is a hit compound obtained by the above-mentioned screening method, on the tumorigenicity of RD cells was evaluated. That is, 6-well cells with RD cells suspended in a basal medium containing 0 ⁇ M, 0.1 ⁇ M, 0.5 ⁇ M, 2 ⁇ M, or 5 ⁇ M tunicamycin so that the number of cells per well is about 5,000. A total of 3 wells were sown on the culture plate. The amount of basal medium per well is 3 mL.
  • FIG. 23 shows a photograph (FIG. 23A) of colony formation by RD cells after culturing in a basal medium containing 0 ⁇ M or 5 ⁇ M tunicamycin for 2 weeks as a typical appearance of experimental results, and RD cells having each concentration.
  • the measurement result (FIG. 23B) of the number of colonies formed after culturing in the basal medium containing tunicamycin for 2 weeks is shown.
  • RD cells cultured in a medium containing 0.1 ⁇ M or more of tunicamycin did not form any observable colonies. This result indicates that tunicamycin has an effect of suppressing the tumorigenicity of RD cells.
  • these compounds can be suitably used as a substance that inhibits translation involving CDKAL1 and further as a substance that suppresses the tumorigenicity of cancer stem cells.
  • a substance that inhibits translation in which CDKAL1 is involved can be efficiently and easily obtained.
  • the above 101 compounds are classified based on the diseases for which the compounds have been conventionally used (in the case of compounds used for a plurality of diseases, the diseases that are the main target of application (FIGS. 24 and 25).
  • the diseases were classified based on the diseases listed on the left side), and the breakdown was 16 types of cardiovascular disease-related drugs, 5 types of endocrine disease-related drugs, 32 types of infectious disease-related drugs, and inflammation / immune diseases. There are 11 related drugs, 8 metabolic disease-related drugs, 24 neurological disease-related drugs, and 5 other drugs.
  • CDKAL1 is involved among FDA-approved compounds that have not been used for cancer treatment in the past. A large number of compounds have been found that have activity that inhibits translation. This result strongly indicates that the translation mechanism involving CDKAL1 is a completely new drug discovery target, and these compounds that strongly inhibit the translation involving CDKAL1 are active ingredients of agents for the treatment of cancer. It has been shown that it can be suitably used.
  • RNA sequencing was performed. Specifically, a sequence common to a group of genes (FIG. 26A, in a trapezoid) whose translation is suppressed by suppressing the expression of CDKAL1 was analyzed using a MEME (Multiple Em for Motif Elicitation) program.
  • MEME Multiple Em for Motif Elicitation
  • GES Guide-enriched sequence
  • CES Cytosine-enriched sequence
  • the identified minimal GES (hereinafter, also referred to as "mGES") is as shown in FIG. 26B, with GGCGGGCGGGCGCGCGCC as the basic sequence, the first G may be A, the second G may be C, and the second G may be C.
  • the third C may be A
  • the fourth G may be A
  • the fifth G may be C
  • the sixth C may be A
  • the seventh G may be A or C
  • the eighth G may be A
  • 9th C may be U or A
  • 10th G may be A or U
  • 11th G may be C
  • 12th C may be A, 13th G. May be U or C or A
  • the 14th G may be U or A or C
  • the 15th C may be G or A or U.
  • the identified minimal CES (hereinafter, also referred to as "mCES") is as shown in FIG. 26B, with GCCGCCGCCGCCGCC as the basic sequence, the first G may be U or C, and the second C may be.
  • the third C may be U
  • the fourth G may be U or C
  • the fifth C may be U
  • the sixth C may be A or U
  • the seventh G may be U.
  • the 8th C may be U
  • the 9th C may be G
  • the 10th G may be U
  • the 11th C may be G
  • the 12th C may be U
  • G may be U
  • 14th C may be U
  • 15th C may be G.
  • mRNA containing mGES or mCES in 5'UTR was subject to translation control by CDKAL1.
  • a base sequence encoding firefly luciferase as a reporter protein, and a base sequence containing four mGES (4 ⁇ mGES) in the 5'UTR on the upstream side, that is, the untranslated region on the 5'end side Alternatively, a plasmid that produces an mRNA having a base sequence containing 4 mCES (4 ⁇ mCES) as a transcript in cells (hereinafter, this plasmid is referred to as “4 ⁇ mGES-5'UTR-firefly luciferase reporter plasmid” and "4 x mCES-5'UTR-firefly luciferase reporter plasmid" was prepared according to a conventional method.
  • the base sequence encoding 4 ⁇ mGES-5'UTR-firefly luciferase in the 4 ⁇ mGES-5'UTR-firefly luciferase reporter plasmid is as shown in SEQ ID NO: 37 in the sequence listing.
  • the base sequence corresponding to the 5'untranslated region containing 4 ⁇ mGES is as shown in SEQ ID NO: 38 in the sequence listing.
  • the base sequence corresponding to mGES is as shown in FIG. 29A.
  • the base sequence encoding 4 ⁇ mCES-5'UTR-firefly luciferase in the 4 ⁇ mCES-5'UTR-firefly luciferase reporter plasmid is as shown in SEQ ID NO: 39 in the sequence listing.
  • the base sequence corresponding to the 5'untranslated region containing 4 ⁇ mCES is as shown in SEQ ID NO: 40 in the sequence listing.
  • the base sequence corresponding to mCES in the base sequence corresponding to the 5'untranslated region containing 4 ⁇ mCES is as shown in FIG. 29B.
  • siRNA siRNA targeting CDKAL1.
  • siRNA siRNA targeting CDKAL1
  • siControl siRNA that does not target any gene as a negative control
  • siRNA siRNA targeting CDKAL1
  • the base sequence of siRNA (siCDKAL1) targeting CDKAL1 is as described in SEQ ID NOs: 41 and 42 in the sequence listing.
  • siRNA Silence Select Negative Control No. 1 siRNA (Thermo Fisher Scientific, Catalog No .: 4390843) (hereinafter, this siRNA is referred to as "siControl”) was used.
  • siCDKAL1 or siControl was introduced into RD cells. That is, RD cells were seeded into a 35 mm culture dish so as to be about 80% confluent. The next day, mix 60 pm Albanyl of siRNA (siCDKAL1 or siControl) with 10 ⁇ L of Lipofectamine TM RNAiMAX Transfection Regent (Thermo Fisher Scientific) and 500 ⁇ L of Opti-MEM. A mixture was prepared, the resulting mixture was added dropwise to the medium, and then incubated in an environment of 37 ° C. and 5% CO 2 for 48 hours.
  • cells are isolated by trypsin treatment, the isolated cells are suspended in a basal medium, and the number of cells per well is 1 ⁇ 10 4 (10,000 cells). Seeded into 96-well plates and incubated for 24 hours.
  • each reporter plasmid was introduced into RD cells by the following procedure. That is, SALL2-5'UTR-firefly luciferase reporter plasmid, GAPDH-5'UTR-firefly luciferase reporter plasmid, ACTB-5'UTR-firefly luciferase reporter plasmid, 4 ⁇ mGES-5 at a dose of 0.1 ⁇ g per well.
  • SALL2-5'UTR-firefly luciferase reporter plasmid GAPDH-5'UTR-firefly luciferase reporter plasmid
  • ACTB-5'UTR-firefly luciferase reporter plasmid 4 ⁇ mGES-5 at a dose of 0.1 ⁇ g per well.
  • the expression of the luciferase reporter having the 5'UTR base sequence of GAPDH and ACTB mRNA is not affected by the knockdown of CDKAL1, whereas the expression of 5'UTR containing mGES or mCES is unaffected.
  • the expression of the luciferase reporter having a base sequence was significantly reduced by knockdown of CDKAL1.
  • the degree of decrease in the expression of the luciferase reporter by knockdown of CDKAL1 was the degree of decrease in the expression of the luciferase reporter in the 5'UTR of SALL2 mRNA, which is a positive control.
  • the expression was as low as or higher than that of the luciferase reporter having a base sequence.
  • This result indicates that mRNA having mGES or mCES in 5'UTR is subject to translational control by the translation mechanism in which CDKAL1 is involved. Even when the number of mGES or mCES contained in the 5'UTR was changed from 4, 2, 6, or 8, a decrease in the expression of the luciferase reporter due to knockdown of CDKAL1 was observed.
  • SEQ ID NO: 1 DNA sequence encoding the first shRNA targeting human CDKAL1 (shCDKAL1 # 1)
  • SEQ ID NO: 2 RNA of shCDKAL1 # 1
  • SEQ ID NO: 3 of the sense strand of siRNA that can arise from shCDKAL1 # 1.
  • RNA SEQ ID NO: 4 RNA sequence number 5 of the antisense strand of siRNA that can result from shCDKAL1 # 1: DNA sequence number 6: shCDKAL1 encoding a second shRNA (shCDKAL1 # 2) that targets human CDKAL1.
  • RNA sequence of # 2 SEQ ID NO: 7 RNA sequence of siRNA sense strand that can result from shCDKAL1 # 2: RNA sequence number 8: RNA sequence of siRNA antisense strand that can result from shCDKAL1 # 2: Targeting human SALL2 RNA sequence of the DNA sequence encoding the first shRNA (shSALL2 # 1) 10: RNA sequence of shSALL2 # 1 11: RNA sequence of the sense strand of siRNA that can be derived from shSALL2 # 1 From shSALL2 # 1 RNA sequence of antisense strand of possible siRNA SEQ ID NO: 13: DNA sequence encoding a second shRNA (shSALL2 # 2) targeting human SALL2 SEQ ID NO: 14: RNA sequence of shSALL2 # 2 SEQ ID NO: 15: shSALL2 RNA sequence number 16 of the sense strand of siRNA that can result from # 2: RNA sequence number 17 of the antisense strand of siRNA that can arise from shSALL2 # 2: first shRNA
  • SEQ ID NO: 33 DNA sequence encoding GAPDH-5'UTR-firefly luciferase
  • SEQ ID NO: 34 Corresponding to the 5'untranslated region of the mRNA of the human GAPDH gene in the DNA sequence encoding the GAPDH-5'UTR-firefly luciferase reporter.
  • DNA sequence to be sequenced SEQ ID NO: 35: DNA sequence encoding ACTB-5'UTR-firefly luciferase
  • SEQ ID NO: 36 5'untranslated human ACTB gene mRNA in the DNA sequence encoding ACTB-5'UTR-firefly luciferase reporter.
  • DNA sequence corresponding to the RNA sequence of the 5'untranslated region of an mRNA SEQ ID NO: 39: 4 ⁇ mCES-5'UTR-DNA sequence encoding the firefly luciferase
  • a new agent for cancer treatment targeting a specific translation mechanism in cancer cells or cancer stem cells is provided. Further, according to the screening method according to another aspect of the present invention, it is possible to efficiently screen a substance that inhibits a specific translation mechanism in cancer cells or cancer stem cells. These agents or substances obtained by the screening method can be expected to be applied as therapeutic agents for cancer, and the industrial applicability of the present invention is great.

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Abstract

La présente invention aborde le problème de la fourniture d'un agent de traitement anticancéreux, ledit agent ciblant la traduction spécifique des cellules cancéreuses ou des cellules souches cancéreuses ou d'un gène exprimé par la traduction et, en conséquence, permettant d'inhiber la prolifération des cellules ou d'éliminer leur caractère souche. Pour résoudre ce problème, un agent de traitement anticancéreux est concerné. Ledit agent comprend, en tant que principe actif, un ingrédient permettant d'inhiber l'expression d'un gène dans la traduction à laquelle participe CDKAL1.
PCT/JP2021/045165 2020-12-09 2021-12-08 Agent de traitement anticancéreux, et procédé de criblage pour son principe actif Ceased WO2022124342A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115287286A (zh) * 2022-06-24 2022-11-04 中国人民解放军军事科学院军事医学研究院 一种长链非编码RNA lnc1267在调节细胞增殖与存活中的应用
CN115475166A (zh) * 2022-09-13 2022-12-16 中南大学湘雅医院 瑞伐拉赞类化合物的应用、药物及制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016060741A (ja) * 2014-09-12 2016-04-25 国立大学法人 鹿児島大学 成人ｔ細胞白血病治療薬
US20160116474A1 (en) * 2013-06-20 2016-04-28 The Broad Institute, Inc. Compositions and methods for detecting and treating glioblastoma

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160116474A1 (en) * 2013-06-20 2016-04-28 The Broad Institute, Inc. Compositions and methods for detecting and treating glioblastoma
JP2016060741A (ja) * 2014-09-12 2016-04-25 国立大学法人 鹿児島大学 成人ｔ細胞白血病治療薬

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DE-FREITAS-JUNIOR JULIO CESAR MADUREIRA, BASTOS LILIAN GOLÇALVES, FREIRE-NETO CARLOS ALBERTO, ROCHER BÁRBARA DU, ABDELHAY ELIANA S: "N-glycan biosynthesis inhibitors induce in vitro anticancer activity in colorectal cancer cells", JOURNAL OF CELLULAR BIOCHEMISTRY JOHN WILEY & SONS, INC., vol. 113, no. 9, 1 September 2012 (2012-09-01), pages 2957 - 2966, XP055941246, ISSN: 0730-2312, DOI: 10.1002/jcb.24173 *
HOU HELEI, GE CHAO, SUN HEFEN, LI HONG, LI JINJUN, TIAN HUA: "Tunicamycin inhibits cell proliferation and migration in hepatocellular carcinoma through suppression ofCD44s and the ERK1/2 pathway", CANCER SCIENCE, vol. 109, no. 4, 1 April 2018 (2018-04-01), JP , pages 1088 - 1100, XP055941250, ISSN: 1347-9032, DOI: 10.1111/cas.13518 *
IRIMURA TATSURO, GONZALEZ ROBERT, NICOLSON2 GARTH L: "Effects of Tunicamycin on B16 Metastatic Melanoma Cell Surface Glycoproteins and Blood-borne Arrest and Survival Properties1", CANCER RESEARCH, vol. 41, no. 9, 1 January 1981 (1981-01-01), pages 3411 - 3418, XP055941251 *
OLSSON A Y; FEBER A; EDWARDS S; TE POELE R; GIDDINGS I; MERSON S; COOPER C S: "Role of E2F3 expression in modulating cellular proliferation rate in human bladder and prostate cancer cells", ONCOGENE, vol. 26, no. 7, 14 August 2006 (2006-08-14), London , pages 1028 - 1037, XP037741984, ISSN: 0950-9232, DOI: 10.1038/sj.onc.1209854 *
TAKESUE YOSHIHIRO, WEI FAN-YAN, FUKUDA HIROYUKI, TANOUE YUKI, YAMAMOTO TAKAHIRO, CHUJO TAKESHI, SHINOJIMA NAOKI, YANO SHIGETOSHI, : "Regulation of growth hormone biosynthesis by Cdk5 regulatory subunit associated protein 1-like 1 (CDKAL1) in pituitary adenomas", ENDOCRINE JOURNAL, vol. 66, no. 9, 1 January 2019 (2019-01-01), JP , pages 807 - 816, XP055941261, ISSN: 0918-8959, DOI: 10.1507/endocrj.EJ18-0536 *
YU DOU, KHAN OMAR F., SUVÀ MARIO L., DONG BIQIN, PANEK WOJCIECH K., XIAO TING, WU MEIJING, HAN YU, AHMED ATIQUE U., BALYASNIKOVA I: "Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 114, no. 30, 25 July 2017 (2017-07-25), pages E6147 - E6156, XP055941242, ISSN: 0027-8424, DOI: 10.1073/pnas.1701911114 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115287286A (zh) * 2022-06-24 2022-11-04 中国人民解放军军事科学院军事医学研究院 一种长链非编码RNA lnc1267在调节细胞增殖与存活中的应用
CN115287286B (zh) * 2022-06-24 2023-03-07 中国人民解放军军事科学院军事医学研究院 一种长链非编码RNA lnc1267在调节细胞增殖与存活中的应用
CN115475166A (zh) * 2022-09-13 2022-12-16 中南大学湘雅医院 瑞伐拉赞类化合物的应用、药物及制备方法
CN115475166B (zh) * 2022-09-13 2023-11-21 中南大学湘雅医院 瑞伐拉赞类化合物的应用、药物及制备方法

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