WO2016159377A1 - Procédé de criblage pour identifier un agent thérapeutique contre le cancer - Google Patents

Procédé de criblage pour identifier un agent thérapeutique contre le cancer Download PDF

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WO2016159377A1
WO2016159377A1 PCT/JP2016/060975 JP2016060975W WO2016159377A1 WO 2016159377 A1 WO2016159377 A1 WO 2016159377A1 JP 2016060975 W JP2016060975 W JP 2016060975W WO 2016159377 A1 WO2016159377 A1 WO 2016159377A1
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expression
ews
atf1
cells
cancer
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泰広 山田
克憲 蝉
山本　拓也
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国立大学法人京都大学
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

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  • the present invention relates to a method for screening for a therapeutic drug for cancer.
  • molecular targeting drugs that is, therapeutic drugs that directly target proteins related to important intracellular signals related to cancer cell growth and progression
  • searches for proteins that can become new drug discovery targets are being actively pursued.
  • target proteins for cancer treatment include proteins encoded by genes that are unique or overexpressed in cancer cells.
  • Non-patent Document 1 a technique for reprogramming somatic cells has been developed and attempts have been made to reinitialize cancer cells. Such attempts have been focused on changing the properties of cancer cells themselves. There are no reports on screening for cancer drugs using cancer cell reprogramming.
  • An object of the present invention is to provide a method for screening for a therapeutic drug for cancer.
  • the present inventors have found that when initializing cancer cells, the target cells (that is, important intracellular signals related to cancer cell growth / progression). Repression of the expression of genes that are known to be related) enhances reprogramming, in other words, the proteins encoded by these target genes maintain their characteristics as cancer cells, And found that it has the effect of suppressing the fate change of cancer cells. Furthermore, the present inventors have found that the enhanced initialization depends on the expression of transposable elements. Therefore, when cancer cells expressing a target protein of an existing molecular target drug were brought into contact with the molecular target drug to suppress the activity of the target protein, it was confirmed that the expression of the transposable element was increased.
  • the test substance when the expression of a transposable element in a cancer cell for which an effective target protein is known is increased under contact with the test substance, it can be confirmed that the test substance has a function of suppressing the activity of the target protein. Became clear.
  • the test substance when targeting any cancer cell, if the test substance increases the expression of the transposable element in the cancer cell, the test substance is a cell involved in the growth / progress of the cancer cell. Since it can be presumed to have a function of suppressing the activity of some protein related to the internal signal, it becomes possible to directly screen candidate substances for cancer therapeutic agents without identifying the target protein (target gene). As a result of further studies based on these findings, the present inventors have completed the present invention.
  • a method for screening for a therapeutic drug for cancer comprising the following steps; (I) a step of measuring the expression level of a transposable element in contact with or non-contact with a test substance in a target cancer cell; and (ii) under non-contact in contact with a test substance. A step of selecting the test substance as a cancer therapeutic agent when the expression level of the transposable element increases in comparison.
  • the transposable element is at least one sequence selected from endogenous retrovirus, LINE, SINE, and fragments thereof.
  • transposable element is at least one sequence selected from HERV-H, HERV-W, L1-ORF2, ERVK and fragments thereof.
  • a method for identifying a protein that can be a drug discovery target of a cancer therapeutic drug comprising the following steps; (I) Expression of a transposable element in a cancer cell containing a gene encoding a test protein in a form in which expression can be controlled under conditions for expressing the gene or under conditions where expression of the gene is suppressed A step of measuring the amount, and (ii) when the expression level of the transposable element is increased under the condition in which the expression of the gene is suppressed as compared with the condition under which the gene is expressed. A process of selecting proteins that can be targets for drug discovery for cancer treatment.
  • the present invention it is not necessary to identify a target gene, and it becomes possible to directly screen for a therapeutic drug for cancer that becomes a molecular target drug.
  • FIG. 1A shows a schematic diagram describing a method for inducing EWS / ATF1 expression and introducing reprogramming factors.
  • FIG. 1B shows a phase contrast microscopic image of sarcoma cell line G1297 after introduction of reprogramming factor when EWS / ATF1 expression was induced (DOXD0.2 ⁇ g / ml) or not (DOX 0 ⁇ g / ml).
  • FIG. 1C shows the results of measuring the number of colonies formed after introduction of reprogramming factor when EWS / ATF1 expression was induced (DOX 0.2 or 0.1 ⁇ g / ml) or not (DOX 0 ⁇ g / ml). .
  • FIG. 1A shows a schematic diagram describing a method for inducing EWS / ATF1 expression and introducing reprogramming factors.
  • FIG. 1B shows a phase contrast microscopic image of sarcoma cell line G1297 after introduction of reprogramming factor when
  • FIG. 1D shows RT-PCR of EWS / ATF1 expression induction by adding 0.2 ⁇ g / ml DOX to iPS cell lines (C-1, C-2, C-3 and C-4) established from sarcoma cell line G1297. The result confirmed by is shown.
  • FIG. 1E shows the results of chromosome microarray (CGH array) analysis in the sarcoma cell line G1297 and an iPS cell line established from the same.
  • FIG. 1F shows the results of measuring the expression of Nanog in the iPS cell lines (C-1, C-2, C-3 and C-4) by RT-PCR (left figure) and staining the iPS cell line with Nanog and DAPI. The stained image (right figure) is shown.
  • FIG. 1G shows a stained image of teratoma formed by transplanting iPS cells established from sarcoma cell line G1297 subcutaneously into nude mice.
  • FIG. 1H shows a photograph of a chimeric mouse generated by injecting iPS cells established from the sarcoma cell line G1297 into a blastocyst.
  • FIG. 1I shows that EWS / ATF1 expression was induced (DOX 0.2 or 0.1 ⁇ g / ml) or not induced (DOX 0 ⁇ g / ml) on day 4 after introduction of reprogramming factor (4F) into sarcoma cell line G1297.
  • FIG. 1J shows that EWS / ATF1 expression was induced on mouse embryonic fibroblasts (MEF) 4 days after introduction of reprogramming factor (4F) (DOX 0.2 or 0.1 ⁇ g / ml) or not (DOX (0 ⁇ g / ml) shows the results of FACS in which the positive rate of SSEA1 was measured 10 days after 4F introduction.
  • FIG. 1K shows 4F introduction or GFP when EWS / ATF1 expression was induced (ON) or not induced (OFF) on day 4 after introduction of reprogramming factor (4F) or GFP into sarcoma cell line G1297.
  • FIG. 1L shows a schematic diagram describing EWS / ATF1 expression induction and a method for introducing MYOD1.
  • Figure 1M shows the results of RT-PCR measurement of Myogenin expression in cells in which MYOD1 was introduced into the sarcoma cell line G1297, with or without EWS / ATF1 expression induction (ON).
  • FIG. 1N shows staining with Myosin heavy chain (MHC) and DAPI, in which MYOD1 was introduced into sarcoma cell line G1297, when EWS / ATF1 expression was induced (ON) or not (OFF).
  • MHC Myosin heavy chain
  • FIG. 1 O shows 6 days after MYOD1 introduction or GFP introduction when EWS / ATF1 expression was induced (ON) or not induced (OFF) on day 4 after introduction of MYOD1 or GFP into sarcoma cell line G1297
  • TSS transcription initiation site
  • FIG. 2B shows ChIP-seq analysis using antibodies against H3K4me3 and H3K27Ac in chromosome 12 with and without EWS / ATF1 expression induced in sarcoma cell line G1297 (ON) or without (OFF) and FAIRE -seq Shows the result of analysis.
  • EWS / ATF1 indicates the binding region of EWS / ATF1, and H3K4me3, H3K27Ac
  • FAIRE indicate the amount of H3K4me3-histone, the amount of H3K27Ac-histone, and the amount of unbound histone, respectively. Shows the peak.
  • FIG. 2C shows the result of ChIP-seq analysis using an antibody against H3K9me3 in the vicinity of the transposable element with or without induction of EWS / ATF1 in the sarcoma cell line G1297.
  • the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read permillion) when EWS / ATF1 is not induced (OFF). Therefore, a high value means that H3K9me3 is enhanced in this region when EWS / ATF1 expression is induced (ON).
  • FIG. 2D shows the expression of L1 (left figure) and MMERVK10c (right figure) with RT-PCR when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297.
  • the measurement results are shown.
  • FIG. 2E shows H3K9me3 in the vicinity of the transcription start site (TSS) of the gene induced by EWS / ATF1 when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297.
  • TSS transcription start site
  • FIG. 2F shows Reduced Representation Bisulfite Sequencing (RRBS at the CpG site in the ERVK region when EWS / ATF1 expression was induced (ON) or not induced (OFF) in ES cells, somatic cells, and sarcoma cell line G1297. ) Shows the result of the analysis.
  • FIG. 3A shows the results of measuring the amount of H3K9me3 at the Oct3 / 4, Nanog and Sox2 binding sites of ES cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3.
  • FIG. 3B shows the results of measuring the amount of H3K9me3 at the MYOD1 binding site of myotube cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3.
  • FIG. 3C shows the results of measuring the amount of Oct3 / 4 at the Oct3 / 4 binding site of ES cells when EWS / ATF1 expression was induced (ON) or not induced (OFF).
  • FIG. 3D shows the introduction of 4F in the sarcoma cell line G1297 with or without induction of EWS / ATF1 expression (ON) or 4 days after introduction of reprogramming factor (4F) or GFP.
  • recovered from the cell of the 6th day after GFP introduction by a microarray is shown.
  • FIG. 3E shows that when EWS / ATF1 expression was induced (ON) or not induced (OFF) in cells introduced with MYOD1 into the sarcoma cell line G1297, expression of EWS / ATF1 was induced (ON), and Suv39h1 was induced by RNAi.
  • FIG. 3F shows the case where EWS / ATF1 expression was induced (ON) on day 4 after introduction of MYOD1 or GFP into the sarcoma cell line G1297, and Suv39h1 expression was suppressed (siSuv39h1) or not (siControl).
  • FIG. 3G shows the gene ontology (GO) term of the gene whose expression increased when Suv39h1 expression was suppressed in the result of FIG. 3F.
  • FIG. 4A shows a schematic diagram describing the method of introduction of reprogramming factor, MYOD1 or GFP and siRNA into the MP-CCS-SY cell line.
  • FIG. 4B shows that when the reprogramming factor (+ 4F) or GFP was introduced into the MP-CCS-SY cell line, expression of EWS / ATF1 was suppressed by RNAi (siEWS / ATF1) or expression was not suppressed (siControl).
  • FIG. 4C shows a schematic diagram describing the introduction of reprogramming factors into HCC827 or SK-BR3 cell lines and the method of adding each drug.
  • 4D shows the case where DMSO, 5FU or Lapatinib (Lap) was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-) Of PODXL expression by RT-PCR (left figure) and when reprogramming factor was introduced into HCC827 cell line (Dox (4F) +) or not (Dox (4F)-)
  • the result (right figure) which measured the expression of PODXL by RT-PCR when DMSO, 5FU, or Gefitinib (Gef) was added in is shown.
  • FIG. 4E shows the results when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-).
  • Results of microarray analysis left figure
  • Gefitinib (Gef) or DMSO when reprogramming factor was introduced into HCC827 cell line Dox (4F) +
  • Dox (4F)- The result (right figure) of the microarray analysis in the case of adding is shown.
  • FIG. 4F shows the results of measuring the expression of L1 OFR2 (left figure) and HERV-W (right figure) by RT-PCR when Lapatinib (Lap) or DMSO was added in the SK-BR3 cell line.
  • FIG. 4G shows the results of measuring the expression of L1 OFR2 (left figure) and HERV-W (right figure) by RT-PCR when Gefitinib (Gef) or DMSO was added in the HCC827 cell line.
  • Fig. 4H shows the results when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line when reprogramming factor was introduced (Dox (4F) +) or not (Dox (4F)-).
  • FIG. 4I shows the results of microarray analysis of the expression of all genes or HERVH-related genes when Lapatinib (Lap) or DMSO was added to the SK-BR3 cell line (left figure) and Gefitinib (Gef) to the HCC827 cell line Or the result (right figure) which performed microarray analysis about the expression of all the genes at the time of DMSO addition or a HERVH related gene is shown.
  • FIG. 5A shows a growth curve when doxycycline (DOX) 0, 0.1, 0.2, 1 or 2 ⁇ g / ml was added to sarcoma cell line G1297 and cultured.
  • DOX doxycycline
  • FIG. 5B shows that when the reprogramming factor was introduced into the sarcoma cell line G1297 (+ 4F) or not (-4F), expression of EWS / ATF1 was induced (ON) or not (OFF) ) Shows the results of measuring the amount of Oct3 / 4 in each case by Western blotting.
  • FIG. 5C shows a phase contrast microscopic image of iPS cells established after introduction of reprogramming factor when EWS / ATF1 expression was not induced (DOX 0 ⁇ g / ml).
  • FIG. 5D shows the results of measuring the expression levels of Oct3 / 4 and Nanog by RT-PCR when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297.
  • ESC ES cell
  • FIG. 5E shows a schematic diagram describing the method of induction of EWS / ATF1 expression and introduction of reprogramming factors.
  • FIG. 5F shows that EWS / ATF1 expression was induced (ON) or not induced (OFF) in cells (+ 4F) or cells not introduced (GFP) into the sarcoma cell line G1297.
  • FIG. 6A shows ChIP using antibodies against H3K4me3 and H3K27Ac at the transcription start site (TSS) of all genes when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297.
  • TSS transcription start site
  • FIG. 6B shows ChIP-seq using an antibody against H3K9me3 in the vicinity of the transcription start site (TSS) of ERVK when EWS / ATF1 expression was induced (ON) or not induced (OFF) in sarcoma cell line G1297.
  • FIG. 6C shows the results of ChIP-seq analysis using an antibody against H3K9me3 in chromosome 15 when EWS / ATF1 expression was induced (ON) or not induced (OFF) in the sarcoma cell line G1297.
  • the transcription region of LTR / ERVK is shown in the figure below.
  • 6D shows the transcription initiation site of the gene induced by EWS / ATF1 in the vicinity of the transposable element when EWS / ATF1 expression was induced (ON) or not (OFF) in the sarcoma cell line G1297.
  • the result of the ChIP-seq analysis (random mapping method) using the antibody with respect to H3K9me3 in (TSS) is shown.
  • the vertical axis shows the logarithm of RPM when EWS / ATF1 expression is induced (ON) with respect to RPM (read permillion) when EWS / ATF1 is not induced (OFF). Therefore, a high value means that H3K9me3 is enhanced in this region when EWS / ATF1 expression is induced (ON).
  • FIG. 7A shows the transcription initiation site (TSS), EWS / ATF1 binding site when EWS / ATF1 expression was induced (ON) or not induced (OFF) in ES cells, somatic cells, and sarcoma cell line G1297. Results of Reduced Representation Bisulfite Sequencing (RRBS) analysis at CpG sites at Oct3 / 4 binding site, Sox2 binding site and Nanog binding site are shown.
  • FIG. 7B shows the results of measuring the amount of H3K9me3 at the Ascl1-binding site of neural stem cells when EWS / ATF1 expression was induced (ON) or not induced (OFF). In the figure, shading indicates the amount of H3K9me3.
  • FIG. 7A shows the transcription initiation site (TSS), EWS / ATF1 binding site when EWS / ATF1 expression was induced (ON) or not induced (OFF) in ES cells, somatic cells, and sarcoma cell line G1297. Results of Reduce
  • FIG. 7C shows ChIP-seq analysis using antibodies against H3K4me3 and H3K27Ac at the Oct3 / 4 binding site of ES cells when EWS / ATF1 expression was induced (ON) or not (OFF) and FAIRE- Results of seq analysis (left figure) and results of ChIP-seq analysis and FAIRE-seq analysis using antibodies against H3K4me3 and H3K27Ac at the MyoD1 binding site of myotube cells (right figure) are shown.
  • FIG. 8A shows a schematic diagram describing a method for inducing EWS / ATF1 expression and introducing a reprogramming factor for ChIP analysis.
  • FIG. 8B shows 6 days after introduction of MyoD1 or GFP when EWS / ATF1 expression was induced (ON) or not induced (OFF) 4 days after introduction of MyoD1 or GFP into sarcoma cell line G1297.
  • recovered from the said cell is shown.
  • FIG. 8C shows the expression of histone modifying enzymes (Suv39h1, Suv39h2, Eed, Suz12, and Ezh2) in the sarcoma cell line G1297 when EWS / ATF1 expression was induced (ON) or not (OFF). The result confirmed by PCR is shown.
  • FIG. 9A is a schematic diagram illustrating the method of induction of expression of EWS / ATF1 for RT-PCR analysis, introduction of MyoD1, and introduction of each siRNA, and the histone modifying enzymes (Suv39h1, Suv39h2, Ezh2, Suz12 and Eed). The effect of expression suppression is shown.
  • FIG. 9A is a schematic diagram illustrating the method of induction of expression of EWS / ATF1 for RT-PCR analysis, introduction of MyoD1, and introduction of each siRNA, and the histone modifying enzymes (Suv39h1, Suv39h2, Ezh2, Suz12 and Eed). The effect of expression suppression is shown.
  • FIG. 9A is a schematic diagram illustrating the method of induction of expression of EWS / ATF1 for RT-PCR analysis, introduction of MyoD1, and introduction of each siRNA, and the histone modifying enzymes (Suv39h1, Suv39h2, Ezh2, Suz12 and Eed).
  • FIG. 9B shows the results obtained by measuring the expression of L1 promoter by RT-PCR when Suv39h1 or Ezh2 expression was suppressed by RNAi in sarcoma cell line G1297.
  • FIG. 9C shows a PiggyBac vector construct for introducing the reprogramming factor and a phase contrast image and a fluorescence image of the cell after introduction into SK-BR3.
  • FIG. 10 shows DMSO, 5FU (1 mM or 10 mM) or Gefitinib (Gef) in the case where the reprogramming factor was introduced into the A549 cell line (Dox (4F) +) or not (Dox (4F) ⁇ ).
  • FIG. 11A shows a schematic diagram describing the method of introduction of the reprogramming factor (4F) by retrovirus and the introduction of anticancer agents (Alectinib, Imatinib) into H2228 and KBM7.
  • FIG. 11A shows a schematic diagram describing the method of introduction of the reprogramming factor (4F) by retrovirus and the introduction of anticancer agents (Alectinib, Imatinib) into H2228 and KBM7.
  • FIG. 12A shows that when reprogramming factors are introduced into H2228 and KBM7, Alectinib (0 ⁇ M (control), 1 ⁇ M or 10 ⁇ M) is added to H2228, and Imatinib (0 ⁇ M (control) is added), 1 ⁇ M or The expression of L1 when 10 ⁇ M) is added is measured by RT-PCR, and the transcription level is normalized by the GAPDH value.
  • FIG. 12A shows that when the expression of EWS / ATF1 was induced in the mouse sarcoma cell line G1297 (DoxON) or not induced (DoxOFF), the reprogramming factor was introduced (+ 4F) or not, respectively.
  • FIG. 12B shows the results of measuring the expression of ERVK in the case of DoxON and DoxOFF by RT-PCR and normalizing the transcription level with ⁇ -actin (Actb) value.
  • the present invention provides a method for screening for a therapeutic agent for cancer, comprising the following steps; (I) a step of measuring the expression of a transposable element in contact with a test substance in a target cancer cell; and (ii) the transposer from a condition in which the cancer cell is not in contact with the test substance. A step of selecting the test substance as a cancer therapeutic agent when the expression of a bull element increases.
  • cancer means a malignant tumor, including carcinoma (malignant tumor derived from epithelial cells), sarcoma, and other leukemias, and is not limited to a specific cancer.
  • the cancer cell used in the present invention is a cell constituting a malignant tumor, and may be a cell line or a cell isolated from a living body.
  • cancer cells used in the present invention are cancer cells (for example, EWS / ATF1 fusion gene forced expression) whose effective target protein is known (ie, known to be sensitive to an existing molecular target drug).
  • cancer cells gefitinib-sensitive mutant EGFR-expressing cancer cells, lapatinib-sensitive HER2-amplified cancer cells, alectinib-sensitive EML4-ALK fusion gene-expressing cancer cells, imatinib-sensitive chronic myeloid leukemia cells, etc.
  • cancer cells whose effective target protein is unknown may be used.
  • test substance in the screening method of the present invention, any test substance can be used, and any known compound and novel compound may be used.
  • the test substance may also be (1) a biological library method, (2) a synthetic library method using deconvolution, (3) “one-bead one-compound” live Can be obtained using any of a number of approaches in combinatorial library methods known in the art, including rally methods, and (4) synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67).
  • Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
  • the contact between the cancer cell and the test substance may be performed by appropriately adding the test substance to the culture solution for cancer cells.
  • the culture solution for cancer cells is used for culturing animal cells.
  • the medium can be prepared as a basal medium. Examples of the basal medium include IMDM medium, Medium ⁇ 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium and the like. Is included.
  • the medium may contain serum or may be serum-free.
  • the medium can be, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules
  • albumin for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, small molecules
  • One or more substances such as compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines and the like may also be included.
  • culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C., preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1 to 10%, preferably about 5%.
  • the O 2 concentration is 1-20%.
  • the O 2 concentration may be 1 to 10%.
  • the contact time between the cancer cell and the test substance is not particularly limited, and examples thereof include 1 hour, 2 hours, 6 hours, 12 hours, 1 day, 1.5 days, 2 days or more.
  • Epigenetic modification is a method of analyzing methylation of DNA by a method of concentrating methylated DNA, a method using base substitution by bisulfite treatment, or a method using a restriction enzyme sensitive to methylation, Perform chromatin immunoprecipitation (ChIP) with modified histone antibodies, analyze the concentrate with RT-PCR or next-generation sequencer (ChIP-seq analysis), extract the open chromatin region on the genome with FAIRE, and generate the next generation Analysis can be performed by a method using a sequencer (FAIRE-seq analysis) or the like.
  • a transposable element means a gene sequence called a mobile genetic element or transposon that is moved or copied on a genome and inserted into the genome.
  • a preferred transposable element used as an index for screening for a therapeutic drug for cancer of the present invention is a retrotransposon.
  • a retrotransposon is a sequence copied to RNA, and thus the copied RNA can be used as an index.
  • retrotransposons include LTR (long terminal repeat) type retrotransposons (endogenous retroviruses) and non-LTR type retrotransposons.
  • the sequence of the transposable element can be obtained from the NCBI database as appropriate. Using this sequence information, the expression level of the transposable element or its fragment can be analyzed by reverse transcriptase PCR or quantitative reverse transcriptase PCR. , Northern blot analysis, immunohistochemistry, array analysis, RNA-seq analysis and combinations thereof. The expression of the transposable element or a fragment thereof can be evaluated by analysis of epigenetic modifications such as DNA methylation and histone modification.
  • the kit for screening for a cancer therapeutic agent includes a primer or a probe used for measuring the expression level of the above-described transposable element.
  • the kit for screening for a therapeutic drug for cancer may further include a document or instructions describing the measurement procedure.
  • it may further include a panel of various cancer cells having different effective target proteins (wherein the effective target protein has the same meaning as described above).
  • a method for identifying a protein that can be a drug discovery target of a cancer therapeutic agent includes the following steps: (I) a transposer under a condition for expressing a candidate gene or a condition in which the expression of the gene is suppressed in a cancer cell containing a gene (candidate gene) encoding a test protein in a form capable of controlling expression.
  • the method includes a step of selecting the test protein as a protein that can be a drug discovery target of a therapeutic drug for cancer.
  • candidate genes are identified as a result of exhaustive analysis of gene expression in cancer cells using a microarray or the like, and are highly expressed in genes or cancer cells that are specifically expressed in cancer cells. Genes that are present.
  • the expression controllable form means a form capable of ON / OFF of the expression of the candidate gene.
  • the candidate gene is an inducible promoter (eg, metallothionein promoter (induced by heavy metal ions), heat shock protein promoter. (Induced by heat shock), Tet-ON / Tet-OFF promoter (induced by addition or removal of tetracycline or its derivative), steroid-responsive promoter (induced by steroid hormone or its derivative), etc.)
  • inducible promoter eg, metallothionein promoter (induced by heavy metal ions), heat shock protein promoter. (Induced by heat shock), Tet-ON / Tet-OFF promoter (induced by addition or removal of tetracycline or its derivative), steroid-responsive promoter (induced by steroid hormone or its derivative), etc.
  • Expression vectors and the like Confirmation / evaluation of expression of the transposable element is the same as the screening method for cancer therapeutic agents described above.
  • the EWS / ATF1 fusion gene described in YamadaYK, et al, J Clin Invest. 123: 600-610, 2013 is induced in a doxycycline (Dox) -dependent manner
  • Dox doxycycline
  • a possible mouse sarcoma cell line also referred to as G1297 strain
  • the sarcoma cells have been confirmed to stop growth in vitro and to regress tumors in vivo due to EWS / ATF1 expression cessation.
  • G1297 was initialized by introducing an initialization factor into G1297.
  • EWS / ATF1 sarcoma cells expressing EWS / ATF1
  • reprogramming factors 4F: Oct3 / 4, Sox2, Klf4, and c-Myc
  • iPS cell-like colonies were confirmed by the expression of 4F (FIGS. 1A to 1C).
  • An iPS cell-like cell line was established by picking up this iPS cell-like colony (FIG. 5C).
  • the induction of iPS cells was performed by the following method.
  • Oct3 / 4 Sox2, Klf4 and c-Myc were each introduced into Plat-E cells using a pMXs-based retroviral vector, and the virus-containing supernatant was collected and filtered through a 0.45 ⁇ m cellulose acetate filter.
  • G1297 was seeded at 8 ⁇ 10 5 cells per 60-mm dish, infected with the virus-containing supernatant, and replaced with LIF-containing ES medium on the third day after infection.
  • the established iPS cell-like cell line was confirmed to express EWS / ATF1 in a doxycycline-dependent manner, similar to the parent sarcoma cell (FIG. 1D). Furthermore, some chromosomal abnormalities were also observed, confirming that the iPS cell-like cell line was derived from sarcoma cells (FIG. 1E). When the expression of pluripotency-related genes such as Nanog and endogenous Oct3 / 4 (Pou5f1) in iPS cell-like cells derived from the sarcoma cells was compared with ES cells, there was no significant difference (Fig. 1F and FIG. 5D). RT-PCR was performed using the following method.
  • the efficiency of initialization of sarcoma cells was 0.06%, which was shown to be lower than the initialization efficiency of MEF.
  • iPS cell-like cells were administered subcutaneously to immunodeficient mice, teratomas were formed, and chimeric mice could be created by injection into blastocysts (FIGS. 1G and 1H).
  • EWS / ATF1 expression-dependent sarcoma cells pluripotent cells can be obtained by reprogramming by suppressing the expression of EWS / ATF1.
  • EWS / ATF1 acts suppressively in the reprogramming of EWS / ATF1 expression-dependent sarcoma cells.
  • EWS / ATF1 does not suppress the generation of SSEA1-positive cells due to reprogramming in MEF (Fig. 1J), so that the expression of EWS / ATF1 results in the suppression of reprogramming specific to sarcoma cells.
  • sarcoma cells when 4F is introduced (4F-sarcoma cells) or sarcoma cells when GFP is introduced as a negative control
  • the microarray analysis was performed for each of the cases where EWS / ATF1 was expressed and not expressed (FIG. 5E).
  • Microarray analysis was performed using Mouse-Gene-1.0-ST-Array (Affymetrix Inc., Santa Clara, USA). All data analysis was performed using GeneSpring GX software program (version 12; Agilent Technologies, Santa Clara, USA).
  • FIG. 1L the influence of EWS / ATF1 expression on skeletal muscle differentiation induction by introduction of MYOD1 was examined.
  • MYOG Myogenin
  • Fig. 1M MYOD1-introduced sarcoma cells
  • MHC myosin heavy chain
  • EWS / ATF1 expression was examined using ChIP-seq analysis and FAIRE-seq analysis (FIG. 2A and FIG. 6A).
  • TSS transcription start site
  • H3K4me3 is an indicator of the activation promoter. No change was observed.
  • H3K27Ac is an indicator of activation enhancer, at the EWS / ATF1 binding site away from TSS but not TSS.
  • EWS / ATF1 was found to increase H3K9me3 in transposable elements (ERVs, LINEs and SINEs) of EWS / ATF1 expression-dependent sarcoma cells (FIGS. 2C, 6B, 6C and 6D). ). Based on this, EWS / ATF1 expression-dependent sarcoma cells suppressed EWS / ATF1 expression and confirmed the expression of the transposable element, confirming that the expression of the transposable elements L1 and MMERVK10c increased. (FIG. 2D).
  • FIG. 9C shows the effects on the reprogramming of EGFR tyrosine kinase inhibitor gefitinib and HER2 tyrosine kinase inhibitor lapatinib.
  • FIG. 4C shows that when lapatinib was treated with 50% inhibitory concentration (IC 50 ) in 4F-introduced SK-BR-3, it promoted the expression of reprogramming marker PODXL (FIG. 4D). The establishment of fully reprogrammed iPS cells was not successful. On the other hand, treatment with 5-fluorouracil (5FU) at an IC 50 concentration did not increase the expression of PODXL (FIG. 4D).
  • IC 50 inhibitory concentration
  • 5FU 5-fluorouracil
  • cancer cells other than HCC827, SK-BR-3 and G1297 can be applied to the screening method of the present invention. That is, evaluation was performed using a known cancer cell type, a non-small cell lung cancer cell line H2228 expressing the EML4-ALK fusion gene, and a chronic myeloid leukemia cell line KBM7.
  • the H2228 cell line is known to be sensitive to Alectinib, an ALK inhibitor
  • the KBM7 cell line is sensitive to Imatinib, a Bcr-Abl tyrosine kinase inhibitor.
  • a reprogramming factor (4F: Oct3 / 4, Sox2, Klf4 and c-Myc) is introduced into H2228 and KBM7 by retrovirus, and Alectinib is added to the culture medium of H2228 to 1 ⁇ M or 10 ⁇ M on the first day of infection.
  • Imatinib was added to the culture medium of KBM7 so as to be 1 ⁇ M or 10 ⁇ M.
  • cancer cells that can be used in the screening method of the present invention are not limited to specific cell types and can be applied to a wide variety of cancer cell types.
  • RNA-seq analysis examined the expression of transposable elements in cells that had stopped (DoxOFF) and cells that continued to contain Dox (DoxON). As a result, as shown in FIG. 12A, an ERVK transposable element was identified, in which the expression was significantly increased by suppressing the expression of EWS / ATF1.
  • RNA-seq analysis was performed using the following method. After extracting total RNA from each cell using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), a library was created using TruSeq Stranded Total RNA with Ribo-Zero Gold LT sample Prep kit (illumina) . After quantification of the concentration using KAPA Library Quantification kits (Nippon Genetics), sequencing was performed with Hiseq 2500 (illumina) using Hiseq PE Rapid Cluster kit v2-HS. The sequence data was analyzed using TopHat software and Cufflinks software, and visualized by IGV (Integrative Genomics Viewer).
  • cancer cells are adaptable to the environment and can therefore survive and continue to progress. After an initial response to a molecular target drug for cancer, the majority of cancers acquire resistance and cause disease recurrence. However, the mechanism by which cancer cells acquire resistance to drugs is not completely understood, but by inhibiting the oncogene signal, the release of slow transcriptional repression through changes in epigenetic modifications. The phenomenon that cancer cells forced to initialize from the outside can acquire adaptability supports the mechanism of resistance acquisition.
  • cancer cells are said to reduce the level of DNA methylation in transposable elements, but the reduction of inhibitory histone markers in transposable elements by the addition of drugs increases the adaptability of cancer cells This is not inconsistent with the above consideration. Adaptation gained through inhibition of oncogene signaling could change the fate of cancer cells to drug resistant cells, a new mechanism for survival of cancer cells when treated with molecularly targeted drugs Is expected to be offered.

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Abstract

Le problème abordé par la présente invention est de pourvoir à un procédé de criblage pour identifier un agent thérapeutique contre le cancer, qui est un médicament ciblant une molécule, capable de cibler l'une quelconque d'un grand nombre de protéines cibles candidates, parmi les protéines cibles candidates sans qu'il soit nécessaire d'identifier une véritable protéine cible. La solution selon l'invention porte sur un procédé de criblage pour identifier un agent thérapeutique contre le cancer, ledit procédé comprenant les étapes suivantes : (i) mesure quantitative de l'expression d'un élément transposable dans une cellule cancéreuse d'intérêt avec ou sans contact de l'élément transposable avec une substance d'essai ; et (ii), quand l'expression quantitative de l'élément transposable en contact avec une substance d'essai augmente par rapport à celle sans contact avec la substance d'essai, sélection de la substance d'essai à titre d'agent thérapeutique contre le cancer.
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