WO2022124342A1

WO2022124342A1 - Agent for cancer therapy, and screening method for active ingredient thereof

Info

Publication number: WO2022124342A1
Application number: PCT/JP2021/045165
Authority: WO
Inventors: 篤史藤村
Original assignee: 国立大学法人岡山大学
Priority date: 2020-12-09
Filing date: 2021-12-08
Publication date: 2022-06-16
Also published as: JPWO2022124342A1

Abstract

The present invention addresses the problem of providing an agent for cancer therapy, said agent targeting translation specific to cancer cells or cancer stem cells or a gene expressed by the translation and, as a result, being capable of inhibiting the proliferation of the cells or eliminating the stemness thereof. To solve this problem, provided is an agent for cancer therapy that comprises, as an active ingredient, an ingredient capable of inhibiting the expression of a gene in the translation of which CDKAL1 participates.

Description

Screening method for agents for cancer treatment and their active ingredients

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. .. Of the above two processes, the factors that control transcription have been better understood due to the development of comprehensive analysis technology for mRNA, which is a transcript, while the factors that control translation have been developed. It cannot be said that the understanding is sufficiently advanced due to some restrictions.

On the other hand, due to recent advances in nucleic acid analysis technology, it has become clear that in the field of cancer research, the expression of many of the factors that control the stem cell nature of cancer stem cells is regulated by translation. 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.

Many compounds that exhibit cell-killing effects by inhibiting the translation of mRNA into protein are known, but all of them non-selectively inhibit intracellular protein synthesis. For example, cycloheximide produced by Streptomyces griseus, a type of bacterium, inhibits protein synthesis, that is, translation by inhibiting the migration of ribosomes present on mRNA during translation. In addition, puromycin, which is one of the antibiotics, enters the ribosome instead of normal aminoacyl-tRNA and inhibits protein synthesis. As described above, existing translation inhibitors have the drawbacks of being highly toxic to normal cells and having teratogenicity because they non-selectively inhibit the translation of proteins.

On the other hand, 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. For example, in pancreatic β cells, it has been reported that when the expression level of CDKAL1 decreases or the activity of CDKAL1 decreases and the thiomethylation rate of lysine tRNA decreases, the insulin secretion amount decreases (Non-Patent Document). 3). However, as far as the present inventors know, 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.

In the process of continuing research efforts to solve the above problems, 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. As mentioned above, 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. In the present invention, it was found that by suppressing the expression of a gene involved in translation by CDKAL1, the stem cell property, tumorigenicity or self-renewal ability of cancer stem cells is lost, and an excellent antitumor effect can be obtained. completed.

That is, 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. According to the findings found by the present inventors, 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.

Further, in another aspect, the present invention solves the above-mentioned problems by providing a method for screening a substance that inhibits translation in which CDKAL1 is involved. In one preferred embodiment, 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'-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, and the 14th G may be U or A. Or C, and 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. Or C, 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, and the 9th C may be G. Well, 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, and the 15th. C may be G.).

Although details will be described later, according to the findings obtained by the present inventors, 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. Has an RNA sequence represented by the above formula 1 or formula 2. It is known that cytosine- and guanine-rich RNA sequences such as the RNA sequence represented by the

above formula

1 or 2 form a complicated secondary structure, and such an RNA sequence is not on the 5'end side. It is considered that the translation of mRNA contained in the translation region requires the involvement of the translation initiation factor complex in which CDKAL1 is involved. That is, according to the nucleic acid construct having the RNA sequence represented by the

above formula

1 or 2 in the upstream untranslated region of the RNA sequence encoding the reporter protein, 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.

That is, in one preferred embodiment, 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. As shown in the experimental examples described later, according to the above screening method, 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.

According to the agent according to one aspect of the present invention, it 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. By suppressing the expression of the genes involved, the growth of cancer stem cells can be suppressed. On the other hand, according to 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.

Cell lysate (Input) of malignant brain tumor cells JK2 infected with a lentiviral vector expressing shRNA targeting CDKAL1, eIF4E, or eIF4G, or a lentiviral vector expressing shRNA that does not target any gene. ) And the composition of the protein contained in the fraction (m7GTP pull-down) recovered from the cell lysate with m7GTP beads is shown in the figure showing the result of analysis by western blotting. It is a figure which shows the sphere formation ability of the malignant brain tumor cell JK2 which was infected with the lentiviral vector expressing shCDKAL1. It is a figure which shows the enzyme activity of CDKAL1 in the malignant brain tumor cell JK2 infected with the lentiviral vector expressing shCDKAL1. 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. .. It is a figure which shows the sphere formation ability and the colony formation ability of the rhabdomyosarcoma cell RD infected with the lentiviral vector expressing shCDKAL1. (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. (A) Decreased sphere-forming ability and knockdown of CDKAL1 in human liver cancer cells Huh-7, HepG2 infected with a lentiviral vector expressing shCDKAL1; (B) in HepG2 in which CDKAL1 was knocked down. It is a figure which shows the decrease of the colonization ability; (C) is the figure which shows the attenuation of the expression of CD133 and CD44 which are liver cancer stem cell markers in Huh-7, HepG2 which knocked down CDKAL1. (A) Decreased sphere-forming ability and knockdown of CDKAL1 in human prostate cancer cells PC3 and LNCaP infected with a lentiviral vector expressing shCDKAL1; (B) in PC3 and LNCaP knocked down by CDKAL1. It is a figure which shows the attenuation of the expression of CD133 and CD44 which are the prostate cancer stem cell markers. (A) It is a figure which shows the decrease of the sphere forming ability and the knockdown of CDKAL1 in the human gastric cancer cells NUGC3, HGC27, MKN45 infected with the lentiviral vector expressing shCDKAL1; (B) NUGC3, MKN45 which knocked down CDKAL1. It is a figure which shows the decrease of the colonization ability in | (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. 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 shCDKAL1. In the rhabdomyosarcoma cell RD, the amount of change in total RNA due to knockdown of CDKAL1 is plotted on the horizontal axis, and the amount of change in RNA contained in the polysome fraction is plotted on the vertical axis. It is a heat map showing a group of genes that receive translational repression by knockdown of CDKAL1 in rhabdomyosarcoma cell RD. (A) The amount of SALL2 protein in the rhombic myoma cell RD infected with the lentiviral vector expressing shCDKAL1 was evaluated by Western blotting, and (B) the amount of SALL2 mRNA was evaluated by RT-PCT. It is a figure which shows the result. (C) It is a figure which shows the sphere formation ability of the rhabdomyosarcoma cell RD infected with the lentiviral vector expressing shSALL2, and (D) the colonization ability. 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; (B) changes in tumor size over time in mice seeded with Control-C2C12 cells and HRas / shp53-C2C12 cells, and the results of tissue staining; (C). ) It is a figure which shows the result of having evaluated the expression level of CDKAL1 in Control-C2C12 cell and HRas / shp53-C2C12 cell by western blotting; It is a figure which shows the decrease of the sphere formation ability of HRas / skip53-C2C12 cells when the expression of CDKAL1 is suppressed by (E) RNAi; (F) is the figure which shows the decrease of the expression of CDKAL1 by RNAi. It is a figure which shows the influence on the proliferation ability and colony formation ability of Control-C2C12 cell and HRas / skip53-C2C12 cell. (A) 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. The results are shown; (D) 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. It is a figure showing the result of knocking down the expression of CDKAL1 by RNAi and evaluating its sphere-forming ability in RD overexpressing; (C) N-linked glycosylation inhibitor Tunamycin, GSK3 inhibitor. It is a figure which shows the result of having evaluated the expression | expression of the protein of SALL2 and the expression level of the mRNA of SALL2 when BIO and CHIR-98014 which are | (D) In m7GTP beads from RD cells obtained from RD cells on which Tunamycin, an N-linked glycosylation inhibitor, BIO and CHIR-98014, which are GSK3 inhibitors, were allowed to act. It is a figure which shows the result of having analyzed the composition of the protein contained in the recovered fraction (m7GTP celldown) by western blotting. It is a conceptual diagram showing the structure of the RNA construct produced from the SALL2-5'UTR-firefly luciferase reporter plasmid, the GAPDH-5'UTR-firefly luciferase reporter plasmid, and the ACTB-5'UTR-firefly luciferase plasmid. .. Since SALL2-5'UTR has a complex secondary structure compared to GAPDH-5'UTR and ACTB-5'UTR, CDKAL1 is involved in the translation of RNA constructs with SALL2-5'UTR. It is considered that the translation initiation factor complex formed in the above is essential. 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. In the figure, 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, and β-ACTIN is ACTB-5'UTR. -The measurement result of the luciferase activity in the RD cell transfected with the firefly luciferase plasmid is shown. It is a figure which shows the result of a screening experiment. Go6983 and tunicamycin were selected as hit compounds. It is a figure which shows the result of having analyzed the expression level of CDKAL1 and SALL2 in RD cells cultured for 24 weeks in the basal medium containing (A) 0 μM or 5 μM tunicamycin or (B) Go6983 by Western blotting. (A) Microscopic photographs of colonization by RD cells after culturing in a basal medium containing 0 μM or 5 μM tunicamycin for 2 weeks, and (B) RD cells in a basal medium containing each concentration of tunicamycin 2 It is a figure which shows the measurement result of the number of colonies formed after culturing for a week. It is a figure which shows the result of a screening experiment. It is a figure which shows the result of a screening experiment. (A) As a result of analyzing the sequence common to the gene group (inside the trapezoid in the figure) whose expression is reduced at the translation level due to the suppression of the expression of CDKAL1 by RNAi by the MEME program, the guanine-rich sequence (GES) and cytosine were obtained. Rich sequences (CES) were identified; (B) GES and diagrams showing minimal GES (mGES) and minimal CES (mCES) identified as core regions of CES; (C) expression of CDKAL1 by RNAi. It is a figure which shows the expression level of the luciferase reporter after transfecting the luciferase reporter which added mGES and mCES to the 5'end side to the knocked-down RD cell. (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. (A) In the DNA sequence encoding the 4 × mGES-5'UTR-firefly luciferase reporter, the DNA sequence corresponding to the RNA sequence in the 5'untranslated region of the transcript mRNA (underlined indicates mGES); ( B) In the DNA sequence encoding the 4 × mCES-5'UTR-firefly luciferase reporter, the DNA sequence corresponding to the RNA sequence in the 5'untranslated region of the transcript mRNA (underlined indicates mCES).

Hereinafter, the present invention will be described in more detail.

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. Is a drug for cancer treatment, which contains the above as an active ingredient.

The present invention states that 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. As mentioned above, in eukaryotes, gene expression is achieved by the process of transcription from DNA to mRNA, followed by translation from the transcript mRNA to protein. Here, 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. According to the findings found by the present inventors, CDKAL1 plays a role in mediating the binding between 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. As shown in the experimental examples described later, in cancer stem cells, the gene group in which translation is initiated by the involvement of the translation initiation factor complex formed by the involvement of CDKAL1 maintains the stem cell property of the cancer stem cells. , Is deeply involved in the maintenance of tumorigenicity or the maintenance of self-renewal ability. Therefore, by suppressing the expression of genes involved in the translation of CDKAL1, cancer can be treated. In addition, the term "CDKAL1" in the present specification means human CDKAL1 unless otherwise specified.

In the present specification, 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. On the other hand, in the present specification, 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. , ZNF516 gene, HLTF gene, HIC1 gene, MAML2 gene, IKZF5 gene, preferably SALL2 gene, SP9 gene, IRF2BPL gene, ZNF276 gene, IFI35 gene, YAP1 gene, MIER1 gene, more preferably SALL2 gene. It can be the SP9 gene, the IRF2BPL gene, the ZNF276 gene, the IFI35 gene, and more preferably the SALL2 gene. According to the findings obtained by the present inventors, 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. It is considered to be deeply involved in the maintenance of stem cell property, self-renewal ability, or tumorigenicity. In one preferred embodiment, 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.

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). That is, 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. For example, it can be said that CDKAL1 is a gene involved in its translation. Incidentally, 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.

Hereinafter, a more specific embodiment of the agent according to one aspect of the present invention will be described. However, 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. Of the gene expression processes leading up to translation into a protein, 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. Based on experiments using mice, it has been reported that 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). ), 3598-3608.), It is considered that even if the expression of CDKAL1 itself is suppressed, the effect on normal cells other than cancer cells is small. Also, in the experimental examples described later, it has been confirmed that the influence of inhibition of the translation mechanism involved in CDKAL1 on normal cells is extremely small.

There is no particular limitation on the component that suppresses the expression of CDKAL1 contained in the agent according to the present invention, but in one preferred embodiment, 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. That is, 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.

Here, 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. Is. When 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.

As used herein, "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. That is, in the present specification, 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. For example, as long as it has RNAi activity against CDKAL1, 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.

On the other hand, 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. Can be suppressed. That is, the shRNA for CDKAL1 can be a hairpin-type RNA containing a base sequence complementary to the mRNA of CDKAL1.

Similar to the siRNA described, 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. As the 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. For example, as long as it has RNAi activity against CDKAL1, 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 (ASO) 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. PLos ONE 16 (1): e0238753. Etc.) may be used. In one preferred embodiment, 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. For example, 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.).

On the other hand, 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. In a preferred embodiment, 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.

In one preferred embodiment, 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. In addition, it may be chemically modified. For example, in order to prevent the degradation of an RNA molecule by a degrading enzyme in a living body, 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). Further, 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.

Further, 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. You can go out. Examples of these 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. , A transfection method using an appropriate delivery means such as an emulsion is exemplified. When the cell to which the RNA molecule is introduced is a part of a living tissue, the 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.

Further, 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. When the number of mRNA that serves as a template for protein synthesis in the cell decreases, the amount of protein encoded by the mRNA decreases. Therefore, according to 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. Needless to say, 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. Such agents include, for example, shRNA, siRNA, antisense nucleic acid or sgRNA for the gene as an active ingredient.

In one preferred embodiment, 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. As already described, 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. 28A (SEQ ID NOs: 11 and 12) or the RNA sequences set forth in FIG. 28B (SEQ ID NOs: 15 and 16). However, the sequence of siRNA that can be used in the present invention is not limited thereto. For example, as long as it has RNAi activity against SALL2, 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.

Further, in one preferred embodiment, 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. For example, as long as it has RNAi activity against SALL2, 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.

Regarding shRNA, siRNA, antisense nucleic acid, and sgRNA, other parts including the introduction method and preparation method thereof are as described above.

On the other hand, 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.

As shown in the experimental examples described later, as a result of diligent research efforts, 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. We found that the amino acid sequence of is playing an important role. In addition, as a result of further diligent research efforts, 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. , And 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. That is, in one preferred embodiment, the component that inhibits translation involving CDKAL1 can be an N-linked glycosylation inhibitor or a GSK3 inhibitor. 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.

Further, in one preferred embodiment, 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.

As described above, according to the findings obtained by the present inventors, 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.

Further, as described above, according to the findings found by the present inventors, the N-linked type of asparagine at the 107th position from the N-terminal of CDKAL1 in the amino acid sequence 1 to 202 from the amino terminal containing the UPF domain of CDKAL1. 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. 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.

As used herein, the term "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. Sci. USA, 1994, 91 (19), 9022-9026.), In vitro virus method (N. Nemoto et al. FEBS Let., 19). , 414 (2), 405-408.), cDNA display method (J. Yamaguchi et al. Nucleic Acids Research, 2009, Vol. 37, Issue 16, e108), TRAP peptide method (Ishizawa, T. It can be obtained by appropriately using known techniques such as of the American Chemical Society 2013, 135, 5433-40.). For example, when the phage display method is used, 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. .. For example, 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. By repeating this series of operations, 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. As the target protein, 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.

As used herein, 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. For example, 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. Here, 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. For example, 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. After preparing the containing vector, 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.

On the other hand, 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.). In the SELEX method, first, 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. 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.

On the other hand, in one preferred embodiment, 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. According to the findings found by the present inventors, such low molecular weight compounds include, for example, Go6983, tunicamycin, Ozanimod, Gramicidin, Lomitapide, Fenticonazole (Nitrate), Asenapine (hydrochloride), Proph. ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｒｔｅｒｏｌａｎｅ、Ｂｅｐｒｉｄｉｌ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、Ｌｅｖｏｍｅｐｒｏｍａｚｉｎｅ、Ｅｓａｘｅｒｅｎｏｎｅ、Ｄｒｏｎｅｄａｒｏｎｅ（Ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｍｏｒｏｌｆｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｄｏｘｙｃｙｃｌｉｎｅ（ｈｙｃｌａｔｅ）、Ｌｏｐｅｒａｍｉｄｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｆｌｕｐｉｒｔｉｎｅ（Ｍａｌｅａｔｅ）、Ｓｕｌｆａｍｅｔｅｒ、Ｒｅｖａｐｒａｚａｎ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｌｅｖｉｄｉｐｉｎｅ、Ｖｉｎｂｕｒｎｉｎｅ、Ｅｔｈａｃｒｉｄｉｎｅ（ｌａｃｔａｔｅ）、Ｌａｍｉｖｕｄｉｎｅ、Ｅｃａｂｅｔ（ｓｏｄｉｕｍ）、Ｐｒａｍｉｐｅｘｏｌｅ（ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅ）、ＡＺＤ７５４５、Ｃｅｆｔｅｚｏｌｅ（ｓｏｄｉｕｍ）、Ｍｅｄｅｔｏｍｉｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｌａｃｔｏｓｅ、Ｃｌｏｐｅｒａｓｔｉｎｅ　ｆｅｎｄｉｚｏａｔｅ、Ｃｙｃｌｏｂｅｎｚａｐｒｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｅｆａｔｈｉａｍｉｄｉｎｅ、Ｌ－Ａｒｇｉｎｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｇｅｓｔｒｉｎｏｎｅ、Ｄｏｒａｖｉｒｉｎｅ、Ｎｏｒｅｐｉｎｅｐｈｒｉｎｅ、Ｃｌｏｍｉｐｈｅｎｅ（ｃｉｔｒａｔｅ）、Ｎｏｍｅｇｅｓｔｒｏｌ　ａｃｅｔａｔｅ、Ｃｉｎａｃａｌｃｅｔ、Ｔａｌｃ、Ａｍｏｘａｐｉｎｅ、Ｃｌｏｄｒｏｎｉｃ　ａｃｉｄ（ｄｉｓｏｄｉｕｍ　ｓａｌｔ）、Ｐｈｅｎｙｌｂｕｔａｚｏｎｅ、Ｕｐａｄａｃｉｔｉｎｉｂ、Ｂａｃｉｔｒａｃｉｎ、Ｃｉｍｅｔｒｏｐｉｕｍ（Ｂｒｏｍｉｄｅ）、Ｄｉｈｙｄｒｏｅｒｇｏｔｏｘｉｎｅ（ｍｅｓｙｌａｔｅ）、Ｂｅｄａｑｕｉｌｉｎｅ（ｆｕｍａｒａｔｅ）、Ｓｏｌｒｉａｍｆｅｔｏｌ、Ｄｅｌａｖｉｒｄｉｎｅ（ｍｅｓｙｌａｔｅ） , Tiratricol, Afoxolaner, Ladypasvir, Dapsone, Oxiconazole nitra ｔｅ、Ａｒｇｉｐｒｅｓｓｉｎ、Ｒｏｎｉｄａｚｏｌｅ、Ｔｉｃｌｏｐｉｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｉｓｏｐｒｏｐａｍｉｄｅ（ｉｏｄｉｄｅ）、Ｍｅｒｏｐｅｎｅｍ（ｔｒｉｈｙｄｒａｔｅ）、Ｄｅｏｘｙｃｈｏｌｉｃ　ａｃｉｄ　ｓｏｄｉｕｍ　ｓａｌｔ、Ｏｘｉｒａｃｅｔａｍ、Ｅｔｈａｃｒｉｄｉｎｅ（ｌａｃｔａｔｅ　ｍｏｎｏｈｙｄｒａｔｅ）、Ａｍｒｉｎｏｎｅ、Ｍｏｘｉｄｅｃｔｉｎ、（Ｓ）－Ｆｌｕｒｂｉｐｒｏｆｅｎ、Ｄｉｐｈｙｌｌｉｎｅ、Ｍｅｔａｐｒｏｔｅｒｅｎｏｌ（ｈｅｍｉｓｕｌｆａｔｅ）、Ｆｉｄａｒｅｓｔａｔ、Ｓｕｌｔａｍｉｃｉｌｌｉｎ（ｔｏｓｙｌａｔｅ）、Ｐｉｐｅｒｏｎｙｌ　ｂｕｔｏｘｉｄｅ、Ｖｅｒａｐａｍｉｌ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｌａｒｏｐｉｐｒａｎｔ、Ｔｅｇａｓｅｒｏｄ（ｍａｌｅａｔｅ）、Ｏｒｎｉｐｒｅｓｓｉｎ、Ｌ－Ｏｒｎｉｔｈｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｐｅｒｐｈｅｎａｚｉｎｅ、Ｎｉｔｒｅｎｄｉｐｉｎｅ、Ｔｏｆｏｇｌｉｆｌｏｚｉｎ（ｈｙｄｒａｔｅ）、Ｓｕｌｆａｍｅｒａｚｉｎｅ、Ｆｏｓｆｌｕｃｏｎａｚｏｌｅ、Ｖｉｔａｍｉｎ　Ｄ２、Ｏｘｙｂｕｐｒｏｃａｉｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、Ｔｒｉｆｌｕｐｒｏｍａｚｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｌｉｂｅｎｄｏｌ、Ｓｕｌｂｕｔｉａｍｉｎｅ、Ｔｏｌｏｘａｔｏｎｅ、Ｅｍａｍｅｃｔｉｎ（Ｂｅｎｚｏａｔｅ）、Ｐｉｍｅｔｈｉｘｅｎｅ　ｍａｌｅａｔｅ、Ｄｒｏｎｅｄａｒｏｎｅ、Ｄｉｈｙｄｒｏｅｒｇｏｃｒｉｓｔｉｎｅ（ｍｅｓｙｌａｔｅ）、Ｒｏｃｕｒｏｎｉｕｍ（Ｂｒｏｍｉｄｅ）、Ｓｕｌｐｉｒｉｄｅ、Ｄｏｂｕｔａｍｉｎｅ　（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｉｌｎｉｄｉｐｉｎｅ、Ｃｙｐｒｏｈｅｐｔａｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｄｉｃｌｏｆｅｎａｃ（ｄｉｅｔｈｙｌａｍｉｎｅ） , Sulfachloropyridazine, Ioxylan, Pinacidil monohydrate, Hydrofantrine chloride, Cyproheptedine (not limited to, but not limited to, chloride). As shown in the experimental examples described later, 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. Therefore, 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. For example, 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.

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 However, basically any cancer can be used as long as the translation mechanism involved in its initiation is activated. By the way, in the present specification, "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. In a preferred embodiment, 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. Is. Glioblastoma includes glioblastoma (glioblastoma), and glioblastoma may be mesenchymal type or proneural type. As shown in the experimental examples described later, 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.

In one preferred embodiment, 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. 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. Pharmaceutically acceptable 1 to be incorporated into agents (eg, sodium edetate, citric acid, etc.), antioxidants (eg, sodium hydrogen sulfite, sodium sulfite, sodium pyrosulfate, etc.) and / or other conventional pharmaceuticals. It may contain seeds or two or more components. That is, in one preferred embodiment, the agent according to the present invention can be provided as a pharmaceutical composition.

Further, 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. For example, oral administration, sublingual administration, intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, local administration and the like are exemplified, but not limited thereto. In addition, it is not necessary to say that 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., and 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.

Also, according to another aspect of the invention, 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, and the 14th G may be U or A. Or C, and 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. Or C, 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, and the 9th C may be G. Well, 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, and the 15th. C may be G.).

According to the findings obtained by the present inventors, 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.

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. In this specification, for example, when a numerical range is expressed by using "-" such as "4 to 8", the upper limit (4) and the lower limit (8) are included unless otherwise specified. It means a numerical range.

In one preferred embodiment, 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. As such an RNA sequence, for example, 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.

In the nucleic acid construct according to one aspect of the present invention, when 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. Here, 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.

On the other hand, 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. Here, 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. On the other hand, when a fluorescence photometer or a fluorescence microscope can be used, a blue fluorescent protein (BFP: Blue Fluorescent Protein), a green fluorescent protein (GFP: Green Fluorescent Protein), a yellow fluorescent protein (YFP: Yellow Fluorescent Protein), and red fluorescence A fluorescent protein such as a protein (RFP: Red Fluorescent Protein) may be used as a reporter protein. If an absorptiometer is available, a color-developing enzyme protein such as β-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. Examples of such 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. However, 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. In particular, 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. There are no particular restrictions on the 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. Specific examples 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.

As described above, 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.
(1) A first RNA sequence encoding a reporter protein, and a second RNA sequence containing the RNA sequence represented by the above formula 1 and / or the RNA sequence represented by the above formula 2 on the 5'end side thereof. 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.

In the screening method according to one aspect of the present invention, 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. By way of examples, linear or cyclic peptides, amino acids, proteins, antibodies, antibody fragments, nucleic acids, sugars, lipids, natural macromolecules, synthetic macromolecules, inorganic compounds, organic compounds, or combinations thereof. It can be suitably used. Further, 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.

Hereinafter, each step of the screening method according to one aspect of the present invention will be described.

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. When the nucleic acid construct is introduced into a cell in which the translation mechanism involved in CDKAL1 is activated, the RNA construct is produced intracellularly. When 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. On the other hand, 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 In the step (1), 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. When 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. By adding a predetermined amount of the solution containing the test substance thus prepared to the cell culture solution, the solution containing the test substance can be brought into contact with the cells. On the other hand, as the solution containing no test substance, for example, 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. As in the case of 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. Here, it is preferable that the amount of the solution containing the test substance added is equal to the amount of the solution containing the test substance.

By the way, from the viewpoint of sufficiently introducing the test substance into the cells and obtaining a measurement result that sufficiently reflects the translation-suppressing effect of the test substance in the step (3) described later, 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 In the step (2), 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. As described above, 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.

Here, 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. For example, when a luminescent enzyme protein such as firefly luciferase, sea urchin luciferase, or marine luciferase is used as the reporter protein, a substrate having the property of emitting light by receiving an enzymatic reaction by the luminescent enzyme protein (for example, luciferin). By using a luciferous luminometer that measures the luciferous intensity, the intensity of the signal derived from the reporter protein can be measured. On the other hand, when a fluorescent protein such as blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP) is used as the reporter protein, the fluorescence intensity emitted by the fluorescent protein is emitted. May be measured using a fluorescent photometer. When a color-developing enzyme protein such as β-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). When the chromogenic enzyme protein is β-galactosidase, 5-bromo-4-chloro-3-indrill-β-D-galactopyranoside, 2-nitrophenyl-β-D-galactopyranoside, fluorescein- By using β-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.

(4) 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. As described above, 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. That is, for a certain test substance, 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. When it is decreased as compared with the intensity, the test substance is a substance having an action of suppressing the translation mechanism in which CDKAL1 is involved.

It is shown that the greater the decrease in signal intensity, the stronger the effect of suppressing the translation mechanism involved in CDKAL1, and the signal derived from the reporter protein in the cells contacted with the solution containing the test substance. It is preferable to select a 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.

The disclosures of all documents cited herein are incorporated herein by reference in their entirety. Also, if this specification is translated into English and contains the singular words "a", "an", and "the", unless the context clearly indicates otherwise. It shall include not only a single but also multiple ones.

Hereinafter, the present invention will be described in more detail with reference to experimental examples, but these experimental examples are merely examples of embodiments of the present invention and do not limit the scope of the present invention at all.

<Experiment 1. Preparation of lentiviral vector>
In order to perform knockdown experiments on CDKAL1, eIF4E, and eIF4G, outside virus vectors expressing shRNA targeting each were prepared according to a conventional method. The procedure is as follows.

First, a pLKO.1 puro-shRNA plasmid expressing shRNA targeting each of CDKAL1, eIF4E, and eIF4G was prepared according to a conventional method. As the DNA sequence encoding the shRNA targeting CDKAL1, 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. Hereinafter, in the present specification, the shRNA encoded by the DNA sequence of SEQ ID NO: 1 in the sequence listing is referred to as “shCDKAL1 # 1”, and the shRNA encoded by the DNA sequence of SEQ ID NO: 5 in the sequence listing is referred to as “shCDKAL1 # 2”. Sometimes. 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.

Next, 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%. For 293FT cells, 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").

Next, in order to obtain a shRNA-expressing lentiviral vector, the plasmid prepared in the above experiment was transfected into 293FT cells. That is, pLKO.1 puro-shRNA plasmid (10 μg) 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 μg) and pMD2.G (addgene, catalog number: 12259) (2.5 μg) were transfected by lipofection method, followed by 37 ° C., 5% CO. Incubated in ₂ environments for 24 hours. After 24 hours of incubation, the medium on the cell culture dish was removed and new basal medium was added. Then, after continuing the culture for another 48 hours, the supernatant of the medium on the cell culture dish was collected, and the obtained supernatant was used as a sterile filtration filter (product name "New Stella Disk", Kurashiki Spinning Co., Ltd., Catalog No .: The cells were filtered through S-2504) to obtain a medium containing a lentiviral vector expressing a shRNA targeting CDKAL1, eIF4E or eIF4G. In the above procedure, 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.

Hereinafter, 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. At the same time, as a control, a outside virus vector expressing shRNA having a scrambled sequence that does not target any gene (hereinafter referred to as “shControl”) was prepared. This lenti-virus vector is called Lenti-shControl.

<Experiment 2. Knockdown of CDKAL1 in malignant brain tumor cell line JK2 and protein expression analysis by Western blotting>
The malignant brain tumor cell line JK2, which is a model of cancer stem cells, was infected with the lentiviral vector prepared in Experiment 1, and CDKAL1, eIF4E, or eIF4G was knocked down, and translation initiation in JK2 cells in which the expression of each was knocked down. The formation of the factor complex was evaluated by Western blotting.
The malignant brain tumor cell line JK2 was donated by Kumamoto University, a national university corporation, and was provided by Takahiro Yamamoto, Atsushi Fujimura, et al. , IScience 21, 42-56, November 22, 2019. 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). Scientific, Catalog No .: 17502001) at final concentration x 1, Epithelial cell growth factor (EGF) (Fuji Film Wako Junyaku Co., Ltd., Catalog No .: 053-07871) at final concentration 20 ng / mL, Basic fibroblast growth The final concentration of factor (bFGF) (Fuji Film Wako Pure Chemical Industries, Ltd., catalog number: 068-05384) is 20 ng / mL, and the final concentration of heparin sodium salt (Sigma-Aldrich, catalog number: H3149-10KU) is 0. The cells were cultured in Neurobasal medium (ThermoFisher Scientific, Catalog No .: 21103049) (hereinafter, this mixed medium may also be referred to as "JK2 basic medium") added to a concentration of 002 mg / mL.

First, 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.

Next, the formation of translation initiation factor complex in JK2 cells in which the expression of eIF4E or eIF4G was knocked down by infection with a lentiviral vector was evaluated by Western blotting. The procedure is shown below.

Four days after seeding of the above-mentioned cells, the cells were collected and an immunoprecipitation buffer was added to obtain a cytolytic solution having a protein concentration of 1 mg / mL. The immunoprecipitation buffer contains 50 mM Tris-HCl pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40 as the basic composition of the buffer, and is a protease inhibitor ( Product names "couple, EDTA-free, protease inhibitor cocktail", Sigma-Aldrich, catalog number: 11873580001), and phosphatase inhibitors (product name "PhosSTOP", Sigma-Aldrich, catalog number: 49864501), respectively. It is used according to the attached document attached to the product, and further prepared by adding an appropriate amount of RNaseA (Sigma-Aldrich, Catalog No .: R6513) so that the concentration in the cell lysate is 100 μg / mL. Incidentally, RNaseA was added for the purpose of removing RNA and promoting the formation of a translation initiation factor complex.

Next, 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 μL of m7GTP beads (product name “Immobilated γ-Aminophenyl-m7GTP (C10-spacer)”, Jena Bioscience, Catalog No .: AC-155S) resuspended in 1 was added and mixed at 4 ° C. for 16 hours. Then, after repeating precipitation of m7GTP beads by centrifugation, removal of supernatant, and resuspension with immunoprecipitation buffer, 2 × SDS sample buffer (120 mM Tris-HCl pH = 6.8, 4) was added to the centrifuged m7GTP beads. % SDS, 20% glycerol, 0.05% bromophenol blue, 40 mM DTT) were added in an amount of 60 μL, and the mixture was incubated at 95 ° C. for 5 minutes. Then, 15 μL of the supernatant obtained by separating the m7GTP beads by centrifugation was subjected to Western blotting. In addition to the supernatant, a cytolytic solution before protein recovery by m7GTP beads was used for Western blotting as an input sample.

The primary antibodies used for the detection of CDKAL1, eIF4E, eIF4A, and eIF4G in Western blotting are CDKAL1 antibodies (product name "CDKAL1 antibody rabbit polyclonal", Proteintech, Catalog No .: 22988-1-, respectively. AP), 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), and the secondary antibody is an HRP-labeled anti-rabbit IgG antibody (product name "Anti-rabbit IgG"). , HRP-linked Antibody ”, Cell Signaling technology, Catalog No .: 7074) 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.

The results of Western blotting are shown in Fig. 1. As shown in FIG. 1, a cytolytic solution of JK2 cells infected with Lenti-shCDKAL1 # 1 (a cytolytic solution before protein recovery by m7GTP beads. In FIG. 1, it corresponds to "Input" and shRNA "CDKAL1". In (see Lane), 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. On the other hand, in the cells infected with Lenti-shCDKAL1 # 1, no decrease in the expression levels of eIF4E, eIF4A and eIF4G other than CDKAL1 was observed. This result indicates that the gene knockdown due to infection with Lenti-shCDKAL1 # 1 is specific to CDKAL1.

On the other hand, the fraction recovered by coprecipitation with m7GTP beads from the cell lysate of the cells infected with each lentiviral vector was analyzed by Western blotting, and the result corresponds to "m7GTP pull-down" on the right side of FIG. Shown in the lane. Here, since m7GTP has a property of binding to eIF4E, normally, coprecipitation by m7GTP beads recovers eIF4E and eIF4A and eIF4G, which are factors that bind to eIF4E, together with eIF4E. Therefore, in cells in which CDKAL1 is not knocked down, bands corresponding to eIF4A and eIF4G are observed together with eIF4E (in FIG. 1, "m7GTP pull-down", shRNA "Cont.", RNaseA "+". See sample.). On the other hand, from the cytolysate of JK2 cells infected with Lenti-shCDKAL1 # 1, the fractions recovered by coprecipitation with m7GTP beads (“m7GTP pull-down” and shRNA “CDKAL1” in FIG. 1). (Refer to the corresponding lane.) Although a strong band corresponding to eIF4E was confirmed, a band corresponding to eIF4A and eIF4G was hardly confirmed. This result indicates that the binding between eIF4A and eIF4G and eIF4E is attenuated in JK2 cells in which CDKAL1 is knocked down.

The above results indicate that 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.

<Experiment 3. Effect of knockdown of CDKAL1 on self-renewal ability of JK2 cells>
In the same procedure as in Experiment 2, malignant brain tumor stem cells JK2 were infected with the lentiviral vector (Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl) prepared in Experiment 1. Four days after Lentiviral vector infection, cells were isolated using trypsin (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., Catalog No .: 204-16935) according to a conventional method, and then JK2 basic at a cell concentration of 1000 cell / mL. Suspended in medium. 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.

As shown in FIG. 2, in JK2 cells in which CDKAL1 was knocked down by infection with lentiviral vectors expressing shCDKAL1 (Lenti-shCDKAL1 # 1 and Lenti-shCDKAL1 # 2), JK2 infected with Lenti-shControl. The number of spheres formed after 1 week of incubation was significantly lower compared to the cells. Since the sphere-forming ability of cancer stem cells is regarded as an index of the self-renewal ability of cancer stem cells, from the above results, the self-renewal ability of malignant brain tumor cells JK2 is lost by suppressing the expression of CDKAL1. You can see that. In other words, the above results indicate that CDKAL1 is an essential factor for maintaining the self-renewal ability of malignant brain tumor cells JK2.

<Experiment 4. Enzyme activity of CDKAL1 in JK2 cells infected with a lentiviral vector expressing shRNA targeting CDKAL1>
Next, the enzymatic activity of CDKAL1 in JK2 cells infected with a lentiviral vector expressing shRNA targeting CDKAL1 was evaluated by the following procedure. That is, the lentiviral vector (Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl) prepared in Experiment 1 was infected with malignant brain tumor stem cell JK2 by the same procedure as in

Experiment

2, and 4 days after the infection. Later, RNA was recovered from the cells according to a conventional method, and the modification rate of lysine tRNA was determined by using the recovered RNA as a template. The analysis was performed according to the method reported in. The results are shown in FIG.

As shown in FIG. 3, in JK2 cells infected with a lentiviral vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2), compared with JK2 cells infected with Lenti-shControl, The modification rate of lysine tRNA was reduced by more than 80%, confirming a significant decrease in the activity of CDKAL1. This result indicates that CDKAL1 is knocked down in JK2 cells infected with a lentiviral vector expressing shCDKAL1.

<Experiment 5. Effect of knockdown of CDKAL1 on stem cell properties of cancer stem cells>
Next, the effect of knockdown of CDKAL1 on the stem cell property of cancer stem cells was investigated using JK2 cells infected with a leasin viral vector expressing shRNA targeting CDKAL1. First, the malignant brain tumor stem cell JK2 was infected with the lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl prepared in Experiment 1 by the same procedure as in Experiment 2. Four days after infection with the lentiviral vector, cells were isolated using trypsin according to conventional methods and suspended in JK2 basal medium. Then, 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.). , Catalog number: 163-20145), and cells were fixed by incubating at room temperature for 15 minutes. After fixation, wash once with PBS, then add 3% bovine serum albumin (Sigma-Aldrich, catalog number: A7030-100G) and 0.05% Triton X-100 (Sigma-Aldrich, catalog number: 11332481001). Blocking was performed by adding the containing PBS and incubating at room temperature for 30 minutes. After blocking, the primary antibody diluted with antibody diluent (3% bovine serum albumin and PBS containing 0.01% Triton X-100) was reacted in a moist environment at 4 ° C. for 16 hours.

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), both of which are PBS containing antibody diluted solution (3% bovine serum albumin and 0.01% Triton X-100) so that the final antibody concentration is 2 μg / mL. ) Was diluted before use.

After the reaction of the primary antibody, 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 μg / mL. After the reaction of the secondary antibody, wash with PBS three times, and apply the encapsulant containing the cell nucleus staining reagent DAPI (product name "DAPI Fluoromount-G" (Southern Biotech, catalog number: 0100-20)) to the product. 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.

As shown in FIG. 4, in JK2 cells infected with Lenti-shControl, the expression of Vimentin, MSI1, Sox2, and Nestin, which are markers indicating the stem cell nature of cancer stem cells, was strongly confirmed, while normal. Almost no expression of SYP or MAP2, which are markers of differentiation into nerve cells, was observed. This result indicates that the malignant brain tumor cell JK2 is a cell having stem cell properties. On the other hand, in JK2 cells infected with a lentiviral vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2), Vimentin, MSI1, Sox2, Nestin, which are stem cell markers of cancer stem cells, On the other hand, strong expression of SYP and MAP2, which are markers for differentiation into normal neurons, was observed. The above results indicate that knocking down the expression of CDKAL1 in malignant brain tumor stem cells JK2 cells causes the loss of cancer stem cell properties of JK2 cells, which in turn causes differentiation into normal neurons. .. That is, it is shown that CDKAL1 is an essential factor for maintaining the stem cell property of cancer stem cells.

<Experiment 6. Effect of knockdown of CDKAL1 on self-renewal ability of RD cells>
In order to investigate the effect of CDKAL1 knockdown on other cancer stem cells, the rhabdomyosarcoma cell line RD was used to evaluate the sphere-forming ability and colony-forming ability of CDKAL1 after knockdown.

First, 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. Four days after seeding the cells, 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.

As shown in FIG. 5A, in RD cells in which CDKAL1 was knocked down by infection with a lentiviral vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2), JK2 infected with Lenti-shControl. Compared with cells, a significant decrease in sphere-forming ability, which is an index of self-renewal ability, was confirmed. From the above results, it is shown that the self-renewal ability is lost by suppressing the expression of CDKAL1 in the rhabdomyosarcoma cell RD as well as in the malignant brain tumor cell JK2. In other words, the above results indicate that 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.

Next, regarding the evaluation of the sphere forming ability, in the same manner as described above, about 500,000 RD cells were suspended in 4 mL of the basal medium, and Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti 1 mL of a lentiviral vector-containing medium containing any of -shControl was added, and a total of 5 mL was inoculated into a 60 mm cell culture dish. Four days after seeding of the cells, the cells were isolated using trypsin (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd., Catalog No .: 204-16935) according to a conventional method, and suspended in a basal medium. 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.

As shown in FIG. 5B, in RD cells in which CDKAL1 was knocked down by infection with a lentiviral vector expressing shCDKAL1 (Lenti-shCDKAL1 # 1 or Lenti-shCDKAL1 # 2), RD infected with Lenti-shControl was used. The number of colonies formed after 2 weeks of incubation was significantly lower compared to the cells. Since the colony-forming ability of cancer stem cells is used as an index of the tumor-forming ability of cancer stem cells, based on the above results, the tumor-forming ability of rhabdomyosarcoma cells RD can be improved by suppressing the expression of CDKAL1. It turns out that it will be lost. In other words, the above results indicate that CDKAL1 is an essential factor for maintaining the self-renewal ability of rhabdomyosarcoma cells RD.

<Experiment 7. Effect of knockdown of CDKAL1 on other cancer cells>
To investigate the effect of CDKAL1 knockdown on other cancer cells, human malignant melanoma cell line A2058, SK-Mel-28, HMV-II, human liver cancer cell line Huh-7, HepG2, human prostate cancer cell Using the strains PC3, LNCaP, human gastric cancer cell lines NUGC3, HGC27, MKN45, and human malignant brain tumor cell lines MGG4, MGG8, MGG18, sphere-forming ability, colony-forming ability, and cancer stem cell marker after knockdown of CDKAL1. The expression level of was evaluated.

The specific procedure is shown below. For human malignant melanoma cells A2058, SK-Mel-28, HMV-II, human liver cancer cells Huh-7, HepG2, human prostate cancer cells PC3, LNCaP, and human gastric cancer cells NUGC3, HGC27, MKN45, RD cells. Instead of using these cells, the lentivirus vector was infected and CDKAL1 was knocked down by the same procedure as in Experiment 6, and the sphere-forming ability and the colony-forming ability were evaluated. 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-). 72 ^TM ), 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).

In addition, 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).

On the other hand, for human malignant brain tumor cells MGG4, MGG8, and MGG18, the procedure was the same as in Experiment 6 except that these cells were used instead of RD cells and a medium having the following composition was used instead of the basal medium. Infected with a lentiviral vector, knocked down CDKAL1, and evaluated sphere-forming ability: Neurobasal ^TM Medium (Thermo Fisher Scientific) + 1 x B-27 ^TM supplement (Thermo Fisher Scientific) Fisher Scientific) + 20 ng / mL EGF (Fuji Film Wako Junyaku Co., Ltd.) + 20 ng / mL bFGF (Fuji Film Wako Junyaku Co., Ltd.) + 10 μg / mL Heparin (Sigma-Aldrich). The human malignant brain tumor cells MGG4, MGG8, and MGG18 used in this experiment were all provided by Dr. Hiroaki Wakimoto of Massachusetts General Hospital.

In addition, for 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. First, 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. 1 mL was added to make a total of 5 mL, and the mixture was seeded in a 60 mm cell culture dish. Four days after seeding the cells, cytolysates were obtained and subjected to Western blotting according to the procedure described in Experiment 2.

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.

The obtained results are shown in FIGS. 6 to 10. As shown in FIGS. 6 to 10, malignant melanoma (FIG. 6), liver cancer (FIG. 7), prostate cancer (FIG. 8), gastric cancer (FIG. 9), and malignant brain tumor (FIG. 10) by knocking down CDKAL1. ), The sphere-forming ability, which is an index of self-renewal ability, and the colony-forming ability, which is an index of tumor-forming ability, were significantly reduced. In addition, by knocking down CDKAL1, 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. The above results strongly suggest that the translation mechanism in which CDKAL1 is involved is essential for the maintenance of cancer stem cell property regardless of the type of cancer.

<Experiment 8. Effect of CDKAL1 knockdown on tumor growth of rhabdomyosarcoma cell line RMS-YM>
Next, in the rhabdomyosarcoma cell line RMS-YM, the effect of knockdown of CDKAL1 on tumor growth was investigated. First, about 500,000 RMS-YM cells were suspended in 4 mL of basal medium, and 1 mL of a lentiviral vector-containing medium containing Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl was added. A total of 5 mL was added and seeded in a 60 mm cell culture dish. Four days after seeding, 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 μL. 100 μL 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). That is, 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. In the following equation, Pi represents pi, Length represents the maximum diameter of the tumor, and Width represents the tumor diameter in the direction perpendicular to Length.

Figure 11 shows the change in tumor size over time. As shown in 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.

<Experiment 9. Identification of genes that are translated and regulated by CDKAL1 in cancer stem cells>
The rhabdomyosarcoma cell line RD (purchased from JCRB cell bank, cell number: JCRB9072) was infected with a lentiviral vector expressing shCDKAL1 by the same procedure as in Experiment 6, and CDKAL1 was knocked down. That is, 1 mL of a lentiviral vector-containing medium containing Lenti-shCDKAL1 # 1, Lenti-shCDKAL1 # 2, or Lenti-shControl was added to a suspension of about 500,000 RD cells in 4 mL of basal medium. A total of 5 mL was seeded in a 60 mm cell culture dish and incubated in an environment of 37 ° C. and 5% CO ₂ for 4 days. Four days after seeding the cells, cytolytic fluid for obtaining a polysome fraction from RD cells was collected according to the following procedure.

First, cycloheximide was added to the cell culture medium on the cell culture dish at a rate of 100 μg / 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. using a SW-41-Ti swing rotor (Beckman Coulter). After ultracentrifugation, a total of 11 equal fractions were fractionated while monitoring absorption at a wavelength of 254 nm. A triax gradient profiling system (manufactured by BioComp) was used to collect each fraction.

Next, RNA was recovered from each fraction by using the TRIzol reagent (Thermo Fisher Scientific, catalog number: 15596018) according to the procedure manual attached to the product. 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.

Here, for a certain RNA, 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 amount of the RNA contained in the RNA and the amount of the RNA contained in the total RNA recovered from the cell lysate of the RD cells infected with the lentivirus vector expressing shControl without the fractionation step. It's a difference. 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.

On the other hand, for a certain RNA, 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. The difference between the amount of RNA and the amount of RNA contained in the polysome fraction recovered from the cell lysate of RD cells infected with a lentivirus vector expressing shControl. For a certain RNA, 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. As shown in FIG. 12, 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.

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. , ZNF621, MEIS2, JUNB, ZNF337, E2F5, ZKSCAN1, EGR1, NKX6-3, ELK4, HES7, ZIC1, PSIP1, RFX5, E2F3, BARX1, OCEL1, MXI1, MYCL, SOX4, RBP1 It was identified as a group of genes subject to translational repression by knockdown. Among them, SALL2, SP9, IRF2BPL, ZNF276, IFI35, YAP1, MIER1, HOXA7, PHF3, LBX2, KLF7, HOXB6, PLAG1, ZNF484, ZNF516, HLTF, HIC1, HIC1, MAML2, IKZF5 Among them, 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.

<Experiment 10. Effect of SALL2 knockdown on self-renewal ability and tumorigenicity of RD cells>
In the same procedure as in Experiment 6, 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. Four days after seeding the cells, 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). Detection was performed by using the Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Catalog No .: 1705062) and the ChemiDoc Touch Imaging System (Bio-Rad Laboratories) according to the respective instructions. The obtained results are shown in FIG. 14A.

As shown in 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. As shown in 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.

<Experiment 11. Effect of knockdown of SALL2 on sphere formation ability and colonization ability of RD cells>
To perform a knockdown experiment on SALL2, a leasin viral vector expressing shRNA targeting SALL2 was prepared according to the procedure described in Experiment 1. Two types of DNA sequences were used as the DNA sequences encoding the shRNA targeting SALL2. The respective DNA sequences are shown in SEQ ID NO: 9 and FIG. 28A of the sequence listing or SEQ ID NO: 13 and FIG. 28B of the sequence listing. The base sequence underlined in FIGS. 28A and 28B is a region encoding an RNA sequence complementary to the target SALL2 mRNA. Hereinafter, in the present specification, the shRNA encoded by the nucleotide sequence of SEQ ID NO: 9 and FIG. 28A in the sequence listing is referred to as “shSALL2 # 1”, and the 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. Further, the lentiviral vectors expressing shSALL2 # 1 or shSALL2 # 2 are referred to as Lenti-shSALL2 # 1 and Lenti-shSALL2 # 2, respectively.

Next, 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.

As shown in FIGS. 14C and 14D, in RD cells in which SALL2 was knocked down by infection with a lentiviral vector expressing shSALL2 (Lenti-shSALL2 # 1 or Lenti-shSALL2 # 2), sphere-forming ability (self). A significant decrease in replication ability (Fig. 14C) and colony formation ability (tumor-forming ability) (Fig. 14D) was confirmed. This result indicates that SALL2 is essential for maintaining the self-renewal ability and tumorigenicity of RD cells, which are rhabdomyosarcoma cells.

<Experiment 12. Effect of knockdown of SALL2 on tumor growth of rhabdomyosarcoma cells RMS-YM>
Next, using the rhabdomyosarcoma cell line RMS-YM, the effect of knockdown of SALL2 on the antitumor effect was investigated. This experiment was carried out in the same procedure as in Experiment 8 except that a lentiviral vector expressing shSALL2 was used instead of the lentiviral vector expressing shCDKAL1. The obtained results are shown in FIG.

As shown in FIG. 15, 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, Lenti-shControl was used. 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.

<Experiment 13. Effect of CDKAL1 on maintenance and proliferation of normal cells>
13.1 Preparation of malignant tumor cell model and normal cell model First, a malignant tumor cell model and a normal cell model were prepared. As a model of malignant tumor cells, HRas / shp53-C2C12 cells obtained by obtaining mouse myoblast line C2C12 (Riken BioResource Research Center, catalog number: RCB0987) by overexpression of HRas and knockdown of p53 were produced. did. The procedure is as follows.

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. Seven days after the infection, 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. On the other hand, as a model of normal cells, 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.

13.2 Evaluation of tumorigenicity The tumorigenicity of HRas / skip53-C2C12 cells and Control-C2C12 cells was evaluated. Specifically, 100 μL of a cell suspension of both cells (corresponding to 10 × 10 ⁶ cells) is injected subcutaneously into a 6-week-old female BALB / c-nu / nu mouse, and then the injection site. Changes in tumor size over time were evaluated according to the report of Wu et al. (Wu W. et al., Clinical Cancer Research 2013 Oct 15; 19 (20): 5699-5710) as in Experiment 8. The obtained results are shown in FIG. 16B.

As shown in FIG. 16B, 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.

13.3 Evaluation of the expression level of CDKAL1 The expression level of CDKAL1 in Control-C2C12 cells and HRas / skip53-C2C12 cells was evaluated by Western blotting. Western blotting was performed according to the procedure shown in Experiment 2. The obtained results are shown in FIG. 16C.

As shown in FIG. 16C, 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.

13.4 Evaluation of self-renewal ability Next, 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.

As 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.

13.5 Effect of knockdown of CDKAL1 on sphere-forming ability Next, by observing changes in sphere-forming ability by knocking down CDKAL1 in HRas / skip53-C2C12 cells, the self-self of HRas / shp53-C2C12 cells. The effect of CDKAL1 on replication ability was investigated. The specific experimental procedure is as shown below.

First, 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. Hereinafter, 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.

As shown in FIG. 16E, a decrease in the expression level of CDKAL1 was observed in HRas / sheep53-C2C12 cells infected with Lenti-mouse shCDKAL1 # 1 and Lenti-mouse shCDKAL1 # 2, and the number of spheres formed was remarkable. A decrease was observed. This showed that the self-renewal ability of HRas / skip53-C2C12 cells was significantly suppressed by suppressing the expression of CDKAL1.

13.6 Effect of CDKAL1 knockdown on colonization ability Next, by observing changes in colonization ability by knocking down CDKAL1 in HRas / skip53-C2C12 cells, the production of HRas / shp53-C2C12 cells was observed. The effect of CDKAL1 on tumor capacity was investigated. The specific experimental procedure is as shown below.

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.

As shown in FIG. 16F, the effect of knockdown of CDKAL1 on cell proliferation and colonization ability was observed in HRas / skip53-C2C12 cells, whereas it was not observed in Control-C2C12 cells. This indicates that 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.

<Experiment 14. Effect of each domain of CDKAL1 on stabilization of translation initiation factor complex>
The primary structure of CDKAL1 is considered to be, for example, as shown in FIG. 17A, where 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. .. In this experiment, 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.

14.1 Preparation of mutant of CDKAL1 The mutant produced is shown in FIG. 17B. 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, and ^{CDKAL1 deltaN deletes} the amino acid sequence on the N-terminal side including the UPF domain. ing. 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 μg), pTomo plasmid (10 μg; 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 μg) 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 μg). 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.

14.2 Effect of 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. By evaluating whether or not the decrease in the stabilizing effect of the translation initiation factor complex involving CDKAL1 is rescued when the virus is suppressed by RNAi, the variant of CDKAL1 can stabilize the translation initiation factor complex. We verified whether it would contribute. The specific procedure is as shown below.

First, 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.

Next, 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. Specifically, 1 mL of 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.

As shown in FIG. 17C, in RD cells overexpressing GFP, when Lenti-shCDKAL1 # 1 was introduced, bands corresponding to eIF4G and eIF4A were not confirmed in the fraction recovered by m7GTP bead precipitation. rice field. On the other hand, in RD cells overexpressing CDKAL1 ^WT , which is a wild-type CDKAL1, bands corresponding to eIF4G and eIF4A were confirmed even when Lenti-shCDKAL1 # 1 was introduced. A band corresponding to the CDKAL1 ^WT detected based on the Myc-tag added to the CDKAL1 ^WT was also confirmed. This result indicates that CDKAL1 ^WT and eIF4G and eIF4A coprecipitated with the m7GTP beads, in other words, the formation of a translation initiation factor complex. That is, overexpression of GFP does not rescue the suppression of the stabilizing effect of the translation initiation factor complex by RNAi of CDKAL1, but overexpression of CDKAL1 ^WT suppresses the stabilizing effect of the translation initiation factor complex. Was rescued.

On the other hand, in the RD cells expressing the CDKAL1 mutants ^{CDKAL1 deltaC} , ^{CDKAL1 deltaTC} , and CDKAL1 ^UPF , as in the case of the RD cells expressing CDKAL1 ^WT , Lenti-sh CDKAL1 # 1 was introduced. In addition, in the fraction recovered by m7GTP bead precipitation, a band corresponding to eIF4G and eIF4A and a band corresponding to the CDKAL1 mutant detected based on the Myc-tag added to the CDKAL1 mutant were also confirmed. .. This result indicates that overexpression of CDKAL1 ^deltaC , ^{CDKAL1 deltaTC} , and CDKAL1 ^UPF rescues suppression of the stabilizing effect of the translation initiation factor complex. That is, it was shown that these mutants of CDKAL1 exert a stabilizing effect on the translation initiation factor complex, similar to the wild-type CDKAL1.

On the other hand, surprisingly, among the mutants of CDKAL1, in the RD cells expressing ^{CDKAL1 deltaN} , when Lenti-shCDKAL1 # 1 was introduced, in the fraction recovered by m7GTP bead precipitation, No band corresponding to eIF4G and eIF4A was confirmed, and no band corresponding to ^{CDKAL1 deltaN} was confirmed. This result indicates that the suppression of the stabilizing effect of the translation initiation factor complex is not rescued by the overexpression of CDKAL1 ^deltaN , that is, the stabilizing effect of the translation initiation factor complex is impaired in ^{CDKAL1 deltaN} . ing.

14.3 Effect of CDKAL1 mutant on sphere-forming ability Next, in RD cells overexpressing wild-type CDKAL1 or CDKAL1 mutant, when CDKAL1 expression is suppressed by RNAi, sphere-forming ability (self-renewal) It was evaluated whether or not the decrease in ability) was rescued. Specifically, Lenti-shCDKAL1 # 1 or Lenti-shControl is infected with wild-type CDKAL1, a mutant of CDKAL1, or RD cells overexpressing GFP by the same procedure as in Experiment 14.2. After that, the number of spheres was measured according to the procedure shown in Experiment 6. In addition, the expression level of SALL2 protein was evaluated by Western blotting according to the procedure shown in Experiment 10. The obtained results are shown in FIG. 17D.

As shown in FIG. 17D, in RD cells expressing CDKAL1 ^deltaC , ^{CDKAL1 deltaTC} , or CDKAL1 ^UPF as CDKAL1 mutants, the sphere-forming ability was maintained even after RNAi of CDKAL1, whereas ^{CDKAL1 deltaN} was expressed. In RD cells, the ability to form spheres was significantly reduced after RNAi of CDKAL1. This result is consistent with the result in FIG. 17C, and means that among the mutants of CDKAL1, only ^{CDKAL1 deltaN} could not rescue the decrease in self-renewal ability of RD cells. In addition, the decrease in the expression level of the protein of SALL2, which is a gene whose translation is regulated by the translation mechanism involved in CDKAL1, was also not rescued by the expression of ^{CDKAL1 deltaN} . The above results show that the stabilizing action of the translation initiation factor complex involving CDKAL1 is from the amino acid sequence at the N-terminal of CDKAL1, specifically, the amino acids 1 to 202 from the N-terminal of CDKAL1 containing the UPF domain. It shows that the constituent areas are important.

<Experiment 15. Effect of post-translational modification on translation initiation factor complex stabilizing effect>
Experiment 14 showed that the region composed of the 1st to 202nd amino acids from the N-terminus of CDKAL1 containing the UPF domain is important for the stabilization of the translation initiation factor complex. Therefore, in this experiment, we tested this hypothesis based on the hypothesis that post-translational modification in the region may affect the effect of CDKAL1 on stabilizing the translation initiation factor complex.

15. Preparation of CDKAL1 mutant library First, in the region composed of amino acids 1 to 202 from the N-terminal of CDKAL1, amino acids that may undergo post-translational modification are obtained from PhosphoSitePlus, and ELM. (Eukaryotic Liner Motif) Search based on the program. The results are shown in FIG. 18A.

Next, 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. To 50 μL of the obtained PCR product, 1 μL of the restriction enzyme DpnI was added and reacted at 37 ° C. for 1 hour to decompose the template DNA. Using 2 μL of the obtained PCR product after the DpnI reaction, 50 μL of E. coli (NEB Table Compentent E. coli, New England BioLabs, Catalog No .: C3040H) was transformed by a heat shock method (42 ° C., 30 seconds). Converted. To the obtained E. coli-containing solution after transformation, 300 μL of SOC medium was added, and the mixture was shake-cultured at 37 ° C. for 30 minutes. The obtained E. coli-containing solution was seeded on an LB plate containing 50 μg / 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 μg / 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.

15. Effect of CDKAL1 mutant on translation initiation factor complex stabilizing effect Using the DNA plasmid encoding each variant of CDKAL1 obtained as described above, a wrench according to the procedure described in Experiment 14. A viral vector was prepared, and the obtained lentiviral vector was used to express the CDKAL1 mutant in RD cells. Further, the RD cells expressing the CDKAL1 mutant were infected with Lenti-shCDKAL1 # 1 to obtain CDKAL1. Knocked down. Then, the number of spheres was measured according to the procedure shown in Experiment 6. The obtained results are shown in FIG. 18B.

As shown in FIG. 18B, among the mutant libraries examined, 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.

15.3 Effects of post-translational modification inhibition on the translation mechanism involving CDKAL1 S18A / S22A, N107Q, and S153A are phosphorylated by GSK3 (Glycogen synthase kinase 3), or by N-linked glycosylation and phosphorylase kinase, respectively. It was a sequence predicted to undergo phosphorylation. Therefore, in order to verify whether or not the translational mechanism in which CDKAL1 is involved is suppressed by inhibiting the post-translational modification of CDKAL1 using each inhibitor, the expression of SALL2 after the inhibitor is applied is expressed. , The level of mRNA production and protein expression.

The specific procedure is shown below. First, RD cells were seeded in a culture dish having a diameter of 35 mm so as to be about 80% confluent. The next day, 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. ) Or 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 μM, 37. Incubated for 24 hours in an environment of ° C. and 5% CO ₂ . Then, RNA and protein were extracted according to a conventional method and subjected to cDNA preparation and Western blotting for quantitative PCR, respectively.

In the above procedure, TRIzol Reagent (Thermo Fisher Scientific, Catalog No .: 15596018) was used for RNA extraction 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. For the preparation of cDNA, 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. On the other hand, Western blotting was performed according to the procedure shown in Experiment 2. The obtained results are shown in FIG. 18C.

As shown in FIG. 18C, when tunicamycin, which is an N-linked glycosylation inhibitor, and BIO or CHIR-98014, which is a GSK3 inhibitor, are allowed to act, changes in the production of SALL2 mRNA are observed. Although not, the expression level of SALL2 protein was significantly reduced. This result indicates that tunicamycin, which is an N-linked glycosylation inhibitor, and BIO or CHIR-98014, which is an inhibitor of phosphorylation by GSK3, have an action of inhibiting the translation mechanism in which CDKAL1 is involved. There is. In contrast, K252a, an inhibitor of phosphorylase kinase phosphorylation, did not affect the level of SALL2 mRNA production or the level of SALL2 protein expression.

15.4 Effect of Post-Translational Modification Inhibition on Translation Initiation Factor Complex Stabilizing Action As described above, CDKAL1-related translational inhibition in RD cells treated with N-linked glycosylation inhibitors and GSK3 inhibitors. Was confirmed. Therefore, in order to investigate in more detail the mechanism by which translation involving CDKAL1 is inhibited, CDKAL1 is involved in the N-linked glycosylation inhibitor tunicamycin and the GSK3 inhibitor BIO or CHIR-98014. The effect on the formation of the translation initiation factor complex was investigated.

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.

As shown in FIG. 18D, when tunicamycin, which is an N-linked glycosylation inhibitor, and BIO or CHIR-98014, which is a GSK3 inhibitor, are allowed to act, eIF4G is used in the fraction recovered by coprecipitation of m7GTP beads. , EIF4A, and CDKAL1 corresponding bands were not observed. From this result, it was shown that the N-linked glycosylation inhibitor and the GSK3 inhibitor significantly suppress the formation of the translation initiation factor complex in which CDKAL1 is involved.

From the above, among the amino acid sequences 1 to 202 from the amino acid terminal of CDKAL1, the post-translational modification of serine at position 18, serine at position 22, argin at position 107, and serine at position 153 is a translation initiation factor complex by CDKAL1. It has been shown to be important for body stabilization. In addition, 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. As described above, 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.

<Experiment 16: Reporter plasmid for measuring the activity of the translation mechanism by CDKAL1>
From the above experiments, it was found that SALL2 is a gene translated by the translation mechanism involved in CDKAL1, but on the other hand, GAPDH (glycer), which is a housekeeping gene, is also found in cells in which CDKAL1 is knocked down. No decrease in the expression of aldehyde phosphate dehydrogenase) or ACTB (β-actin) at the protein level was observed (the Western blotting analysis shown in FIG. 14 for the fact that the expression level of GAPDH did not change at the protein level. Please refer to the result.). That is, 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. In general, in translations that require the formation of a translation initiation factor complex, 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. By using an 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.

First, an 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.

In addition, as a control, a base sequence encoding firefly luciferase as a reporter protein and a base of 5'UTR of GAPDH or β-actin (ACTB) mRNA as an untranslated region on the upstream side, that is, on the 5'end side thereof. 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. On the other hand, 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.

Next, in order to verify whether the expression of the reporter protein in the cells into which each reporter plasmid was introduced reflects the expression level of CDKAL1 in the cells, 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.

More specifically, in the same procedure as in Experiment 6, 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. Was suspended in a basal medium, seeded in a 35 mm culture dish, and cultured to a cell density of about 80% confluent. There, 2 μg of SALL2-5'UTR-firefly luciferase reporter plasmid, GAPDH-5'UTR-firefly luciferase reporter plasmid, or ACTB-5'UTR-firefly luciferase reporter plasmid, and efficiency of introducing the plasmid into cells. As an index internal standard, 2 μg 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.

Twenty-four hours after transfection, cells are isolated by treatment with trypsin according to a conventional method, suspended in basal medium to prepare a cell suspension, and then the cell suspension is subjected to the number of cells per well. The seeds were sown on a 96-well plate so that the number of cells was 10,000. Twenty-four hours after seeding, the luciferase activity in the cells to which each test subject compound was added was measured. For the measurement of luciferase activity, use 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. The obtained results are shown in FIG. In addition, in FIG. 20, 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.

As shown in FIG. 20, knockdown of CDKAL1 expression in RD cells into which the SALL2-5'UTR-firefly luciferase reporter plasmid has been introduced reduces the luciferase activity, which is a value that reflects the expression level of firefly luciferase in the cells. It was confirmed that In contrast, the GAPDH-5'UTR-firefly luciferase reporter plasmid or ACTB-5, which produces mRNA having the 5'UTR base sequence of GAPDH or ACTB instead of the 5'UTR base sequence of SALL2. In RD cells into which the'UTR-firefly luciferase reporter plasmid was introduced, no change in luciferase activity was observed due to knockdown of CDKAL1 expression. These results indicate that the luciferase activity in cells into which the SALL2-5'UTR-firefly luciferase reporter plasmid has been introduced reflects the activity of the translational mechanism in which CDKAL1 is involved.

<Experiment 17: Screening for substances that inhibit the translation mechanism by CDKAL1>
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. There, 4 μg of SALL2-5'UTR-firefly luciferase reporter plasmid and 2 μg 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. The mixing ratio and procedure of each reagent followed the instructions attached to the product. Twenty-four hours after transfection, cells were isolated by treatment with trypsin according to conventional methods and suspended in basal medium to a cell concentration of 10,000 cells / 100 μL to prepare a cell suspension. Then, the cell suspension was seeded in a 96-well plate so that 100 μL per well, that is, the number of cells per well was 10,000. Twenty-four hours after seeding, each of the plurality of test target compounds to be screened was dissolved in DMSO and diluted with a basal medium so that the concentration of the test substance was 20 μM, and the final concentration of the test substance was 10 μM. 100 μL was added to each well as described above. As a control, a total of 100 μL of a mixed solution of 0.1 μL of DMSO as a control substance and 99.9 μL of basal medium was added to the cell culture medium. Twenty-four hours after the addition of the test target compound, the 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. For the measurement of 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.

Using the luciferase activity in cells to which DMSO was added as a reference (luciferase activity 100%), the degree of decrease in luciferase activity in cells to which each test target compound was added at a concentration of 10 μM was evaluated. The result is shown in FIG. As a result of the screening, it was confirmed that the strongest decrease in luciferase activity was confirmed in the cells to which Go6983 was added among the total of 50 test target substances targeted for screening (FIG. 21A, arrow corresponds to Go6983). It was also confirmed that the luciferase activity was reduced by about 30% in the cells to which tunicamycin was added (FIG. 21B). As described above, as a result of screening, Go6983 and tunicamycin were selected as hit compounds.

<Experiment 18: Translation suppression effect of CDKAL1 by hit compound>
Next, it was examined whether or not the hit compounds Go6983 and tunicamycin obtained in the screening experiment of Experiment 17 suppress the translation mechanism in which CDKAL1 is involved. That is, after RD cells were cultured for 24 hours in a basal medium containing Go6983 or tunicamycin, respectively, for 24 hours, a cell lysate of RD cells was obtained and subjected to Western blotting according to the procedure described in Experiment 2. .. The result is shown in FIG.

As shown in FIG. 22, in the RD cells cultured in the medium containing Go6983 or tunicamycin, the intensity of the band corresponding to SALL2 was clarified as compared with the RD cells cultured in the medium containing the control substance DMSO. A decrease was observed. This result indicates that the expression level of SALL2 is reduced at the protein level in cells in contact with Go6983 or tunicamycin. On the other hand, for the same cells, the intensity of the band corresponding to CDKAL1 did not change even when compared with RD cells cultured in a medium containing DMSO as a control substance. The result is that Go6983 or tunicamycin reduces the expression level of SALL2 without changing the expression level of CDKAL1, that is, the expression level of SALL2 is reduced by inhibiting the translation mechanism in which CDKAL1 is involved. Is shown.

In addition, the results in FIG. 22 suggest that 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 μM, 0.1 μM, 0.5 μM, 2 μM, or 5 μM 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. 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. FIG. 23 shows a photograph (FIG. 23A) of colony formation by RD cells after culturing in a basal medium containing 0 μM or 5 μM 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.

As shown in FIG. 23, RD cells cultured in a medium containing 0.1 μM 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.

From the above results, it is possible to select a substance that suppresses translation involving CDKAL1 by a screening method using a SALL2-5'UTR-firefly luciferase reporter plasmid, and the screening method is a translation involving CDKAL1. It has been shown that it is suitably used as a method for screening a substance that suppresses the tumorigenicity of cancer stem cells.

<Experiment 19. Screening for substances that inhibit the translational mechanism involved in CDKAL1-Part 2->
A compound library of 2203 compounds approved by the FDA (purchased from MedChemExpress) was used to further screen for translation-inhibiting substances involving CDKAL1. The screening procedure is the same as that shown in Experiment 17, except that the compound to be screened is changed.

As a result of the screening, 101 compounds were identified as compounds having an inhibitory effect equal to or higher than that of CDKAL1 RNAi. The names of the identified 101 compounds and the relative activity values of CDKAL1 shown by each compound are shown in FIGS. 24 and 25. In addition, in FIG. 24 and FIG. 25, the activity value of CDKAL1 when treated with each compound is shown as a relative activity value (%) when the activity value when treated with DMSO which is a negative control is 100%. rice field. As shown in FIGS. 24 and 25, 101 compounds obtained by screening reduced the relative activity value of CDKAL1 to the same level as or higher than that of tunicamycin and Go6983. This result indicates that the above 101 compounds suppress the translation involving CDKAL1. That is, 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. As described above, according to the screening method according to one embodiment of the present invention, a substance that inhibits translation in which CDKAL1 is involved can be efficiently and easily obtained.

In addition, 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). In the "Classification" column, 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. Surprisingly, 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.

<Experiment 20. Identification of the base sequence common to 5'UTR of mRNA whose translation is regulated by CDKAL1>
In order to identify the base sequence common to 5'UTR of the mRNA of the gene group whose expression is regulated at the translation level by CDKAL1, the total RNA and the RNA of the heavy fraction after the polysome fraction obtained in Experiment 9 are 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. As a result, GES (Guanine-enriched sequence) and CES (Cytosine-enriched sequence) shown in FIG. 26A were identified as sequences common to the gene clusters whose translation was suppressed by suppressing the expression of CDKAL1. Further analysis by the MEME program to extract the identified GES and CES core regions identified the minimal GES and minimal CES shown in FIG. 26B, respectively, as the core regions of GES and CES.

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, and the eighth G. 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, and the 15th C may be G or A or U.

On the other hand, 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, and 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, and the 13th. G may be U, 14th C may be U, and 15th C may be G.

Next, it was verified whether or not mRNA containing mGES or mCES in 5'UTR is subject to translation control by CDKAL1. Specifically, 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. In the base sequence corresponding to the 5'untranslated region containing 4 × mGES, the base sequence corresponding to mGES is as shown in FIG. 29A.

On the other hand, 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.

Next, in order to verify whether the expression of the reporter protein in the cells into which each reporter plasmid was introduced reflects the expression level of CDKAL1 in the cells, by transfecting siRNA (siCDKAL1) targeting CDKAL1. Each reporter plasmid is transfected into RD cells in which the expression of CDKAL1 is suppressed, or RD cells transfected with siRNA (siControl) that does not target any gene as a negative control, and the reporter protein in each cell. The expression level of was evaluated. The base sequence of siRNA (siCDKAL1) targeting CDKAL1 is as described in SEQ ID NOs: 41 and 42 in the sequence listing. On the other hand, as a negative control, Silence Select Negative Control No. 1 siRNA (Thermo Fisher Scientific, Catalog No .: 4390843) (hereinafter, this siRNA is referred to as "siControl") was used.

The specific experimental procedure is as follows. First, 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 pmоl of siRNA (siCDKAL1 or siControl) with 10 μL of Lipofectamine ^TM RNAiMAX Transfection Regent (Thermo Fisher Scientific) and 500 μL 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. Then, according to a conventional method, 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.

After that, 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 μg per well. As an internal standard for the efficiency of introduction of the'UTR-firefly luciferase reporter plasmid or the 4 × mCES-5'UTR-firefly luciferase reporter plasmid into cells, pGL. 4.74 [hRLuc / TK] vector sea shiitake luciferase reporter plasmid (Promega, Catalog No .: E6921) was transfected. The plasmid was transfected using a commercially available lipofection reagent (TransIT-LT1 Reagent, Takara Bio Inc., catalog number: MIR2300) according to the procedure described in the instruction manual attached to the product. Luciferase activity in each cell was measured 24 hours after plasmid transfection. The measurement of luciferase activity is described in the manual attached to each product using a Dual-Glo Luciferase Assay System (Promega, catalog number: E2940) and a luminometer (product name "MicroLumat Plus LB96V", BERTHOLD). I followed the procedure. The obtained results are shown in FIG. 26C.

As shown in FIG. 26C, 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. In addition, in cells into which a luciferase reporter having a 5'UTR base sequence containing mGES or mCES was introduced, 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.

[Description of sequence listing]
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 (mouse shCDKAL1 # 1) that targets CDKAL1 in mice. ) Encoding DNA sequence No. 18: DNA sequence encoding a second shRNA (mouse shCDKAL1 # 2) targeting mouse CDKAL1 SEQ ID NO: 19: Human CDKAL1 (CDKAL1 ^WT ) amino acid sequence No. 20: RNA sequence number 21 encoding human CDKAL1 (CDKAL1 ^WT ): amino acid sequence number 22 of human CDKAL1 variant ( ^{CDKAL1 6CA} ): DNA sequence number encoding human CDKAL1 variant ( ^{CDKAL1 6CA} ) 23: Amino acid sequence of human CDKAL1 variant ( ^{CDKAL1 deltaC} ) SEQ ID NO: 24: DNA sequence encoding human CDKAL1 variant ( ^{CDKAL1 deltaC} ) SEQ ID NO: 25: Human CDKAL1 variant (CD) KAL1 ^deltaTC ) amino acid sequence SEQ ID NO: 26: DNA sequence encoding a human CDKAL1 variant ( ^{CDKAL1 deltaTC} ) SEQ ID NO: 27: Human CDKAL1 variant (CDKAL1 ^UPF ) amino acid sequence SEQ ID NO: 28: Human CDKAL1 DNA sequence encoding the variant (CDKAL1 ^UPF ) SEQ ID NO: 29: Amino acid sequence of the human CDKAL1 variant ( ^{CDKAL1 deltaN} ) SEQ ID NO: 30: DNA sequence encoding the human CDKAL1 variant ( ^{CDKAL1 deltaN} ) 31 : DNA sequence encoding SALL2-5'UTR-firefly luciferase SEQ ID NO: 32: DNA sequence corresponding to the 5'untranslated region of the mRNA of the human SALL2 gene in the DNA sequence encoding SALL2-5'UTR-firefly luciferase reporter. 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 region SEQ ID NO: 37: 4 x mGES-5'UTR-Firefly Luciferase in the DNA sequence encoding the 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 SEQ ID NO: 40: 4 × mCES-5'UTR-firefly luciferase reporter DNA sequence corresponding to the RNA sequence of the 5'untranslated region of the transcript mRNA in the DNA sequence encoding SEQ ID NO: 41: RNA sequence of the sense strand of siRNA (siCDKAL1) targeting human CDKAL1.
SEQ ID NO: 42: RNA sequence of the antisense strand of siRNA (siCDKAL1) targeting human CDKAL1.

According to the agent according to one aspect of the present invention, 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.

Claims

An agent for cancer treatment that contains as an active ingredient a component that suppresses the expression of genes involved in the translation of CDKAL1.
The agent according to claim 1, wherein the component is a component that suppresses the expression of CDKAL1.
The agent according to claim 2, wherein the component is a vector expressing siRNA, shRNA, antisense nucleic acid or sgRNA against CDKAL1, or the siRNA, SHRNA, antisense nucleic acid or sgRNA.
The agent according to claim 1, wherein the component is a component that reduces mRNA of the gene.
The agent according to claim 4, wherein the component is a vector expressing siRNA, shRNA, antisense nucleic acid or sgRNA for the gene, or the siRNA, shRNA, antisense nucleic acid or sgRNA.
The agent according to any one of claims 1 to 5, wherein the gene is a SALL2 gene.
The agent according to claim 1, wherein the component is a component that inhibits the translation of the gene from mRNA to protein.
The agent according to claim 7, wherein the component is a GSK3 inhibitor and / or an N-linked glycosylation inhibitor.
The agent according to claim 7, wherein the component is a peptide, antibody, antibody fragment, or aptamer that specifically binds to CDKAL1.
The agent according to claim 9, wherein the component specifically binds to the amino acid sequence 1 to 202 from the amino terminus of CDKAL1.
The component is the amino acid sequence 1 to 202 from the amino terminus of CDKAL1 and is characterized by specifically binding to the amino acid sequence containing one or more post-translational modifications of the following (1) to (4). The agent according to claim 9:
(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.
前記成分が、Ｇｏ６９８３、ｔｕｎｉｃａｍｙｃｉｎ、Ｏｚａｎｉｍｏｄ、Ｇｒａｍｉｃｉｄｉｎ、Ｌｏｍｉｔａｐｉｄｅ、Ｆｅｎｔｉｃｏｎａｚｏｌｅ（Ｎｉｔｒａｔｅ）、Ａｓｅｎａｐｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｐｒｏｐａｆｅｎｏｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｓｅｒｔｒａｌｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｒｔｅｒｏｌａｎｅ、Ｂｅｐｒｉｄｉｌ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、Ｌｅｖｏｍｅｐｒｏｍａｚｉｎｅ、Ｅｓａｘｅｒｅｎｏｎｅ、Ｄｒｏｎｅｄａｒｏｎｅ（Ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｍｏｒｏｌｆｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｄｏｘｙｃｙｃｌｉｎｅ（ｈｙｃｌａｔｅ）、Ｌｏｐｅｒａｍｉｄｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｆｌｕｐｉｒｔｉｎｅ（Ｍａｌｅａｔｅ）、Ｓｕｌｆａｍｅｔｅｒ、Ｒｅｖａｐｒａｚａｎ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｌｅｖｉｄｉｐｉｎｅ、Ｖｉｎｂｕｒｎｉｎｅ、Ｅｔｈａｃｒｉｄｉｎｅ（ｌａｃｔａｔｅ）、Ｌａｍｉｖｕｄｉｎｅ、Ｅｃａｂｅｔ（ｓｏｄｉｕｍ）、Ｐｒａｍｉｐｅｘｏｌｅ（ｄｉｈｙｄｒｏｃｈｌｏｒｉｄｅ）、ＡＺＤ７５４５、Ｃｅｆｔｅｚｏｌｅ（ｓｏｄｉｕｍ）、Ｍｅｄｅｔｏｍｉｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｌａｃｔｏｓｅ、Ｃｌｏｐｅｒａｓｔｉｎｅ　ｆｅｎｄｉｚｏａｔｅ、Ｃｙｃｌｏｂｅｎｚａｐｒｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｅｆａｔｈｉａｍｉｄｉｎｅ、Ｌ－Ａｒｇｉｎｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｇｅｓｔｒｉｎｏｎｅ、Ｄｏｒａｖｉｒｉｎｅ、Ｎｏｒｅｐｉｎｅｐｈｒｉｎｅ、Ｃｌｏｍｉｐｈｅｎｅ（ｃｉｔｒａｔｅ）、Ｎｏｍｅｇｅｓｔｒｏｌ　ａｃｅｔａｔｅ、Ｃｉｎａｃａｌｃｅｔ、Ｔａｌｃ、Ａｍｏｘａｐｉｎｅ、Ｃｌｏｄｒｏｎｉｃ　ａｃｉｄ（ｄｉｓｏｄｉｕｍ　ｓａｌｔ）、Ｐｈｅｎｙｌｂｕｔａｚｏｎｅ、Ｕｐａｄａｃｉｔｉｎｉｂ、Ｂａｃｉｔｒａｃｉｎ、Ｃｉｍｅｔｒｏｐｉｕｍ（Ｂｒｏｍｉｄｅ）、Ｄｉｈｙｄｒｏｅｒｇｏｔｏｘｉｎｅ（ｍｅｓｙｌａｔｅ）、Ｂｅｄａｑｕｉｌｉｎｅ（ｆｕｍａｒａｔｅ）、Ｓｏｌｒｉａｍｆｅｔｏｌ、Ｄｅｌａｖｉｒｄｉｎｅ（ｍｅｓｙｌａｔｅ）、Ｔｉｒａｔｒｉｃｏｌ、Ａｆｏｘｏｌａｎｅｒ、Ｌｅｄｉｐａｓｖｉｒ、Ｄａｐｓｏｎｅ、Ｏｘｉｃｏｎａｚｏｌｅ　ｎｉｔｒａｔｅ、Ａｒｇｉｐｒｅｓｓｉｎ、Ｒｏｎｉｄａｚｏｌｅ、Ｔｉｃｌｏｐｉｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｉｓｏｐｒｏｐａｍｉｄｅ（ｉｏｄｉｄｅ）、Ｍｅｒｏｐｅｎｅｍ（ｔｒｉｈｙｄｒａｔｅ）、Ｄｅｏｘｙｃｈｏｌｉｃ　ａｃｉｄ　ｓｏｄｉｕｍ　ｓａｌｔ、Ｏｘｉｒａｃｅｔａｍ、Ｅｔｈａｃｒｉｄｉｎｅ（ｌａｃｔａｔｅ　ｍｏｎｏｈｙｄｒａｔｅ）、Ａｍｒｉｎｏｎｅ、Ｍｏｘｉｄｅｃｔｉｎ、（Ｓ）－Ｆｌｕｒｂｉｐｒｏｆｅｎ、Ｄｉｐｈｙｌｌｉｎｅ、Ｍｅｔａｐｒｏｔｅｒｅｎｏｌ（ｈｅｍｉｓｕｌｆａｔｅ）、Ｆｉｄａｒｅｓｔａｔ、Ｓｕｌｔａｍｉｃｉｌｌｉｎ（ｔｏｓｙｌａｔｅ）、Ｐｉｐｅｒｏｎｙｌ　ｂｕｔｏｘｉｄｅ、Ｖｅｒａｐａｍｉｌ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｌａｒｏｐｉｐｒａｎｔ、Ｔｅｇａｓｅｒｏｄ（ｍａｌｅａｔｅ）、Ｏｒｎｉｐｒｅｓｓｉｎ、Ｌ－Ｏｒｎｉｔｈｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｐｅｒｐｈｅｎａｚｉｎｅ、Ｎｉｔｒｅｎｄｉｐｉｎｅ、Ｔｏｆｏｇｌｉｆｌｏｚｉｎ（ｈｙｄｒａｔｅ）、Ｓｕｌｆａｍｅｒａｚｉｎｅ、Ｆｏｓｆｌｕｃｏｎａｚｏｌｅ、Ｖｉｔａｍｉｎ　Ｄ２、Ｏｘｙｂｕｐｒｏｃａｉｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、Ｔｒｉｆｌｕｐｒｏｍａｚｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ａｌｉｂｅｎｄｏｌ、Ｓｕｌｂｕｔｉａｍｉｎｅ、Ｔｏｌｏｘａｔｏｎｅ、Ｅｍａｍｅｃｔｉｎ（Ｂｅｎｚｏａｔｅ）、Ｐｉｍｅｔｈｉｘｅｎｅ　ｍａｌｅａｔｅ、Ｄｒｏｎｅｄａｒｏｎｅ、Ｄｉｈｙｄｒｏｅｒｇｏｃｒｉｓｔｉｎｅ（ｍｅｓｙｌａｔｅ）、Ｒｏｃｕｒｏｎｉｕｍ（Ｂｒｏｍｉｄｅ）、Ｓｕｌｐｉｒｉｄｅ、Ｄｏｂｕｔａｍｉｎｅ　（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｃｉｌｎｉｄｉｐｉｎｅ、Ｃｙｐｒｏｈｅｐｔａｄｉｎｅ（ｈｙｄｒｏｃｈｌｏｒｉｄｅ）、Ｄｉｃｌｏｆｅｎａｃ（ｄｉｅｔｈｙｌａｍｉｎｅ）、Ｓｕｌｆａｃｈｌｏｒｏｐｙｒｉｄａｚｉｎｅ、Ｉｏｘｉｌａｎ、Ｐｉｎａｃｉｄｉｌ The agent according to claim 7, wherein the agent is one or more selected from the group consisting of monohydrate, hydrochloride, chloride, and Cyproheptazine (hydrochloride sequihydrate).
The agent according to any one of claims 1 to 12, wherein the cancer to be treated is rhabdomyosarcoma or malignant brain tumor.
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, and the 14th G may be U or A. Or C, and 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. Or C, 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, and the 9th C may be G. Well, 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, and the 15th. C may be G.).
The agent according to claim 13, wherein the second RNA sequence is an RNA sequence in the 5'untranslated region of mRNA which is a transcript of the SALL2 gene.
A vector containing the nucleic acid construct according to claim 14 or 15.
A cell containing the nucleic acid construct according to claim 14 or 15.
A method for screening a substance that inhibits translation involving CDKAL1 in cells.
(1) A step of introducing the nucleic acid construct according to claim 14 or 15 into the 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.
A screening method comprising.