JP5995163B2 - Molecular target for MYCN amplified disease and use thereof - Google Patents

Description

  The present specification relates to molecular targets for MYCN gene-amplified diseases and uses thereof.

  Amplification of the MYCN gene is thought to be involved in many diseases. An example is neuroblastoma. Neuroblastoma is known as the most common solid tumor in children. The origin is said to be neural crest cells, and is said to be cancerous in the process of differentiation into sympathetic nerves. Although the cause of the occurrence has not been completely elucidated, amplification of MYCN gene, mutation of ALK, and the like have been reported (Non-patent Document 1). In addition, MYCN amplification type neuroblastoma has a very poor prognosis, and an effective treatment method has not been obtained. MYCN transgenic mice have been established as model mice for neuroblastoma, and there are reports that hemizygotes are closer to human neuroblastoma.

N Engl J Med 362 (23) 2002-2011

  However, when the expression of the MYCN gene is directly suppressed by knockdown or the like, it affects not only cancer cells but also normal cells. Therefore, it is considered that a drug that acts directly on the MYCN gene may cause side effects.

  “Synthetic lethality” is a life phenomenon that results in death only when there is an abnormality in both of the combinations of two genes. “Synthetic lethality” is a concept born from genetics such as yeast, but the present inventors have focused on application to diseases such as cancer. That is, from the viewpoint of synthetic lethality, we considered that there is a possibility of controlling cell viability by combining genetic abnormalities that characterize the pathology and control of other genes. “Synthetic lethality” is useful for searching for new genes and molecular targets that are targets for treatment. Furthermore, if such other genes can be detected, it is considered that treatment that acts only on pathological cells with high selectivity can be achieved by avoiding direct action on genes that characterize pathological conditions.

  This specification pays attention to the gene which shows a synthetic lethal phenotype in relation to the amplification of the MYCN gene, and provides the molecular target and marker for MYCN gene-amplified disease targeting these genes and their use.

  The present inventors use a mouse neuroblastoma model in which the MYCN gene is amplified, a gene whose expression level increases with the progress of canceration, a gene showing a correlation between the patient prognosis and the expression level, MYCN-amplified cells In addition, we searched for various genes that are highly expressed in, and also found synthetic lethal genes that were selectively lethal in MYCN-amplified cells by knocking down these genes. Based on such knowledge, the disclosure of the present specification provides the following means.

  The present specification provides a method for screening molecular targets for diseases caused by genetic abnormalities. This screening method comprises a step of screening one or more other genes whose expression level is increased in a gene abnormal cell presenting a gene abnormality, and the expression of the one or more other genes in the gene abnormal cell. And evaluating one or more other genes using as an index the selective lethality to the abnormal gene cell due to suppression.

  The present specification also provides a method for screening drugs for MYCN-amplified diseases. This screening method is one or more selected from the group consisting of Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein, which are subunit proteins constituting the condensin 1 complex. A step of evaluating one or more test compounds using as an index the decrease in the activity of the protein or suppression of expression of genes encoding these proteins.

  The present specification also provides a method for screening drugs for MYCN-amplified diseases. This screening method comprises a step of supplying one or more test compounds to MYCN-amplified cells, and Smc2 protein, Smc4 protein, CAP- which are subunit proteins constituting the condensin 1 complex in the MYCN-amplified cells. The above 1 or 2 using as an index the decrease in the activity of one or more proteins selected from the group consisting of D2 protein, CAP-H protein and CAP-G protein or suppression of the expression of genes encoding these proteins And a step of evaluating the above test compound.

  The present specification also provides an agent for a MYCN amplified disease. This drug is one or more selected from the group consisting of Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein, which are subunit proteins constituting the condensin 1 complex. A compound that decreases the expression product activity of a gene encoding one or more proteins selected from the group or a compound that suppresses the expression of a gene encoding these proteins can be used as an active ingredient.

  The present specification also provides a prognostic marker in MYCN amplified disease. This prognostic marker is one or more selected from the group consisting of Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein, which are subunit proteins constituting the condensin 1 complex. The expression product of the gene encoding

  The present specification also provides a method for predicting the prognosis of patients with MYCN-amplified disease. According to this prediction method, a test sample collected from a patient with MYCN-amplified disease consists of Smc2, Smc4, CAP-D2, CAP-H, and CAP-G proteins that constitute a condensin 1 complex. The prognosis of the patient can be predicted using the expression status of a gene encoding one or more proteins selected from the group as an index.

It is a figure which shows the relationship between MYCN gene amplification and prognosis in a human neuroblastoma patient. It is a figure which shows the subunit protein in a condensin 1 complex and a condensin 2 complex. It is a figure which shows the evaluation result of the expression level of Smc2 in a hyperplasia and a tumor ganglion in a wild type mouse (2 weeks old) and a MYCN gene transfer mouse (homozygote and hemizygote, 2 and 4 weeks old). It is a figure which shows the evaluation result of the expression level of Smc2 in a hyperplastic ganglion in a wild-type mouse (2 weeks old) and a MYCN transgenic mouse (hemizygote, 2 weeks old). It is a figure which shows the result of having evaluated the expression level of Smc2 using the human neuroblastoma cell line (MYCN gene multicopy and single copy). It is a figure which shows the result of having evaluated the expression level of Smc2 using the human neuroblastoma cell line (MYCN gene single copy). It is a figure which shows the count result of the cell number in the culture | cultivation period of the human neuroblastoma cell line (MYCN gene multi-copy and a single copy) which is and is not Smc2 knocked down. It is a figure which shows the result of the TUNEL assay of Smc2 knockdown IMR32 cell line. It is a figure which shows the result of the TUNEL assay (time-dependent change) of Smc2 knockdown IMR32 cell line. It is a figure which shows the result of having observed the DNA damage in a Smc2 knockdown IMR32 cell line (MYCN gene multicopy) and its non-knockdown line | wire by fluorescent immunostaining. It is a figure which shows the result of having observed the DNA damage in a Smc2 knockdown SK-N-AS cell line (MYCN gene single copy) and its non-knockdown line | wire by fluorescent immunostaining. It is a figure which shows combining the DNA damage in a Smc2 knockdown IMR32 cell line (MYCN gene multiple copy), a Smc2 knockdown SK-N-AS cell line (MYCN gene single copy), and each of these non-knockdown lines. It is a figure which shows the observation result of apoptosis when knocking down Smc2 after supplying two types of DNA damage reagents (cisplatin and camptothecin) and causing DNA damage in the MYCN gene single copy cell line (SH-EP). . It is a figure which shows the ontology of the molecular group which shows a correlation with the expression of Smc2 in a neuroblastoma clinical specimen. It is a figure which shows the expression-analysis result of a Smc2 knockdown MYCN gene amplification cell line (IMR32 cell). It is a figure which shows the expression-analysis result of a Smc2 knockdown MYCN gene single copy cell line (SK-N-AS cell). It is a figure which shows the proliferation mechanism of the MYCN gene amplification cell. It is a figure which shows the proliferation machine of a MYCN gene single copy cell. It is a figure which shows the relationship between Smc2 expression regarding a MYCN amplification type patient and a MYCN non-amplification type patient in a neuroblastoma patient (event-free survival group) It is a figure which shows the relationship between Smc2 expression regarding a MYCN amplification type patient and a MYCN non-amplification type patient in a neuroblastoma patient (all survival group). It is a figure which shows the expression profile of the subunit protein of the condensin 1 complex which contains Smc2 as a subunit, and the subunit protein of another condensin 2 complex regarding a MYCN amplification type patient and a MYCN non-amplification type patient in a neuroblastoma patient. . HcapD2, hCAP-H and hCAP-D3 and hCAP-H2 constituting the condensin 2 complex, which are the subunit proteins constituting the condensin 1 complex in MYCN-amplified patients and non-MYCN-amplified patients in neuroblastoma patients, and these It is a table | surface which shows the p value of the expression level and prognosis of Smc4 common to a composite_body | complex.

  The present specification relates to molecular targets and markers for MYCN gene-amplified diseases and their use.

  According to the disclosure of the present specification, condensin-1 complex is used as a gene group that acts synthetically and deadly on the amplification of MYCN gene in cells that are the cause of MYCN-amplified disease using the concept of synthetic lethality. The body constituent protein gene group was identified. That is, in MYCN-amplified cells, MYCN-amplified cells can be killed by suppressing the expression of any of these gene groups and reducing the activity of these gene products.

  From this, such a gene group or its expression product, condensin 1 complex, or a subunit protein thereof can be used as a molecular target of a drug for MYCN-amplified disease. None of these agents for genes or proteins act directly on the MYCN gene, but act on these molecular targets in MYCN-amplified cells. Therefore, it becomes a drug that acts selectively on MYCN-amplified cells.

  Moreover, the condensin 1 complex which is such a gene group or its expression product can be used as a prognostic marker in MYCN amplification type disease.

  Hereinafter, various aspects of the disclosure of this specification will be described in detail.

(Screening method of molecular targets for diseases caused by genetic abnormalities)
The molecular target screening method disclosed herein is a molecular target screening method for diseases caused by genetic abnormalities. This screening method comprises a step of screening one or more genes whose expression level is increased in a gene abnormal cell presenting the gene abnormality, and suppressing the expression of the one or more genes in the gene abnormal cell And the step of evaluating the one or more genes using as an index the selective lethality against the abnormal gene cell. According to this method, it is possible to screen a molecular target (gene or an expression product thereof) that selectively and synthetically acts on a gene abnormality that causes a disease state. By using such a selective and synthetic lethal gene or an expression product thereof as a molecular target, it becomes possible to screen for drugs that selectively act only on abnormal cells in which a genetic abnormality has occurred.

  Diseases caused by genetic abnormalities include various diseases including cancer. The disease here includes not only individual cancers such as various cancers such as breast cancer, uterine cancer, stomach cancer and lung cancer, but also general cancer diseases including various cancers. This is because a genetic abnormality unique to each cancer disease may occur, or a genetic abnormality shared by a plurality of cancer diseases may occur. For example, it is known that neuroblastoma is caused by an abnormality (amplification) of the MYCN gene. Breast cancer is known to be caused by abnormalities (mutations) in the BRCA1 gene. Information on gene abnormality in a disease can be appropriately obtained by a person skilled in the art from known literature. Examples of gene abnormalities include not only increased expression due to mutation or translocation of specific genes, but also abnormalities such as decreased expression and expression of mutant proteins.

  The gene abnormal cell is sufficient if it includes cells having the gene abnormality. Examples of such genetically abnormal cells include cells collected from patients suffering from the above diseases, cultured cells in addition to collected tissues, cells in which the genetic abnormality has been genetically engineered, cultured cells thereof, and the like. . Furthermore, non-human animals such as mice and zebrafish that have genetically engineered the above-mentioned gene abnormality, or cells and tissues collected from these non-human animals can be mentioned. A person skilled in the art can produce a non-human animal in which a genetic abnormality has occurred, if necessary.

  This screening method can comprise a step of screening one or more genes whose expression levels are increased in the genetically abnormal cells. According to this process, genes that may contribute to the survival of such abnormal gene cells can be screened. In order to screen such genes, it is conceivable to analyze the expression of nucleic acid (DNA or RNA) samples obtained from genetically abnormal cells. A person skilled in the art can analyze a gene having an increased expression level using a known technique. For example, in addition to expression analysis based on the amount of mRNA, multiple copy number analysis of a specific gene based on DNA mutation, translocation in the chromosome, etc. may be combined. Moreover, you may analyze the quantity of possible expressed protein.

  In the screening in this step, one or more other genes whose expression levels tend to increase are screened. In the next step, a plurality of other genes are preferably screened for further screening.

  The screening method further includes the one or more other genes as an index of selective lethality to the gene abnormal cells by suppressing the expression of the one or more other genes in the gene abnormal cells. The process of evaluating can be provided. According to this process, by suppressing the expression from one or more other genes, it is possible to screen for other genes that selectively act on the abnormal gene cells and cause death. This other gene and / or its protein can be a lethal molecular target that is highly selective for abnormal cells.

  A compound that reduces the expression level of another gene that acts selectively and synthetically in combination with an abnormality of a specific gene, a compound (inhibitor) that reduces the activity of the protein that is the product of the other gene, Can be a drug for diseases caused by abnormalities.

(Drug screening method 1 for MYCN amplified disease)
The method for screening a drug disclosed in the present specification is a method for screening a drug for a MYCN-amplified disease, which is a subunit protein constituting a condensin 1 complex, Smc2 protein, Smc4 protein, CAP-D2 protein , One or more test compounds, using as an index the decrease in the activity of one or more proteins selected from the group consisting of CAP-H protein and CAP-G protein or suppression of the expression of genes encoding these proteins The process of evaluating can be provided. The present inventors have found that these proteins or their genes act in a synthetic lethal manner in MYCN-amplified diseases. According to this screening method, compounds that reduce the activity of these proteins as molecular targets or compounds that can suppress gene expression can be screened. Since these compounds do not act directly on the MYCN gene, they can be drugs that avoid or suppress adverse effects on normal cells. A typical example of the MYCN-amplified disease is neuroblastoma. The screened drug becomes a useful drug or a candidate compound thereof for the prevention or treatment of MYCN amplified disease.

    In addition, those skilled in the art can obtain these proteins and genes encoding them in public databases regarding humans and non-human mammals such as mice. For example, in Entrez Nuccleotide (http://www.ncbi.nlm.nih.gov/nucleotide/) on the NCBI website, human smc2 is NM_001042550, NM_001042551, NM_006444, AF092563, AF113673, AJ420415, AK001485, AK292599, AL833191. , BC130385, BN000163, and the like. Similarly, human smc4 is NM_001002800, NM_005496, AB019987, AF092564, AK122939, AK128143, AK315070, AL136877, AL833949, BC146370, BC148796, NM_005496, AB019987, AF092564, AK122939, AK128143, AK315070, AL136877, 370335, BC 146 Can be obtained by referring to the above. Similarly, human CAP-D2 can be obtained by referring to NM_014865, AK022511, AK125155, AK128354, AK303310, BC016913, BC028182, D63880, and the like. Similarly, human CAP-G can be obtained by referring to NM_022346, XM_001058724, XM_223468, AB013299, AF235023, AF331796, AK022512, AK023147, BC068467, BC101476, and the like. Similarly, human CAP-H can be obtained by referring to NM_015341, AK303725, BC024211, CR592757, CR610329, CR620375, D38553, and the like.

  As shown in FIG. 2, the condensin 1 complex plays an important role in chromosome concentration at the cell division (M phase). The condensin 1 complex is composed of subunits whose amino acid sequences are highly conserved from bacteria to humans. The subunits of the condensin 1 complex include Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein.

  The proteins and genes used for evaluation are preferably one or more selected from the group consisting of subunit proteins of these condensin 1 complexes or genes encoding them. Among these proteins, Smc2 is a protein that is amplified in MYCN-amplified cells, and is synthetically lethal by suppressing the activity of the protein (suppression of expression). In addition, it has been found that the subunit protein of the condensin 1 complex as a whole is amplified in MYCN-amplified cells. It is because it becomes clear that it becomes synthetic lethal as well.

  Preferably, the molecular target used for evaluation is more preferably one or more of Smc2 protein, Smc4 protein and CAP-D2 protein, or one or more of genes encoding these proteins. This is because these proteins are particularly relevant to the prognosis of MYCN-amplified diseases such as neuroblastoma. More preferably, it is a Smc2 protein or a gene encoding this protein.

  In this screening method, as a specific method, one or more of the above proteins and one or more test compounds are brought into contact with each other, and the decrease in the activity of the above protein is measured, for example, as ATPase It can be detected by a decrease in enzyme activity. Moreover, the method of evaluating by the unwinding effect | action of the DNA double strand of a condensin 1 complex, etc. is also mentioned. The condensin 1 complex is brought into contact with the test compound, and the decrease or disappearance of the unwinding action of the condensin 1 complex is because the test compound decreases the activity of the subunit protein constituting the condensin 1 complex. . The decrease in the unwinding action of the condensin 1 complex can be detected by the mobility of agarose electrophoresis.

  In addition, one or two or more test compounds are supplied to a cell expressing one or more of the genes encoding the proteins (preferably a cell in which the expression of these genes is enhanced). Examples of methods for evaluating the suppression of the expression of these genes are the amount of expression products (mRNA and protein) and the modulation of the cell cycle.

  A cell in which expression of a gene encoding one or more of the subunit proteins of the condensin 1 complex is enhanced is a useful cell for drug screening for a MYCN-amplified disease. Those skilled in the art can easily obtain such transformed cells by a known genetic engineering technique. As host cells, non-human cells can be used in addition to human cells, and yeast or the like may be used.

  Depending on whether the screening method is an in vivo system or an in vitro system, one or more test compounds may act on the protein or gene in any of these systems. If it is a compound, it will not specifically limit. As appropriate, it may be selected from a compound library, or may be an anti-RNA or siRNA designed to suppress the expression of the gene.

(Method 2 of screening for drugs for MYCN amplified disease)
The drug screening method disclosed in the present specification is a drug screening method for MYCN-amplified diseases, comprising supplying one or more test compounds to MYCN-amplified cells, and the MYCN amplification. Decrease in the activity of one or more proteins selected from the group consisting of Smc2, Smc4, CAP-D2, CAP-H and CAP-G, which are subunits constituting the condensin 1 complex, in cells And the step of evaluating the one or more test compounds, using as an index the suppression of expression of a gene encoding the protein. According to this screening method, a compound that acts more selectively on MYCN-amplified cells can be obtained by evaluating the decrease in the activity of the protein or suppression of the expression of the gene encoding the protein using MYCN-amplified cells. Can be screened. A typical example of the MYCN-amplified disease is neuroblastoma. The screened drug becomes a more effective drug or candidate compound for the prevention or treatment of MYCN-amplified disease.

  Also in this screening method, the embodiment applied in the above-described drug screening method can be applied. That is, the previous drug screening method can be applied to the protein used for evaluation, the evaluation method, and the like. In addition, in this screening method, it is limited to the aspect which supplies a test compound to a cell.

  MYCN-amplified cells are cells with enhanced expression of the MYCN gene. Examples of MYCN-amplified cells include cells collected from patients with MYCN-amplified diseases or cultured cells thereof. Further, it may be a cell engineered using human cells or non-human cells as a host. Furthermore, cells in which the expression of a gene encoding one or more of the subunit proteins of the condensin 1 complex is enhanced while the MYCN gene is amplified can also be used as MYCN amplified cells. A cell in which expression of a gene encoding one or more of MYCN and condensin 1 complex subunit is enhanced is a useful cell for drug screening for MYCN-amplified disease. It has also been found that, when genetically engineered MYCN-amplified cells are produced, the expression of one of the subunits, the Smc2 protein, may be enhanced at the same time. Those skilled in the art can easily obtain transformed cells in which the expression of other subunit proteins including the gene encoding the MYCN gene and the Smc2 protein is enhanced by known genetic engineering techniques. As host cells, non-human cells can be used in addition to human cells, and yeast or the like may be used.

  In addition, according to this specification, the screening method of the chemical | medical agent in BRCA1 and / or BRCA2 amplification type disease (henceforth only BRCA1 amplification type disease) is also provided. Abnormal BRCA1 and / or BRCA2 amplification is considered to be a cause of breast cancer and the like. In BRCA1 and / or BRCA2-amplified cells, it is known that suppression of PARP (poly ADP ribose polymerase) (typically PARP-1) gene expression and Parp protein activity decrease (inhibition) act synthetically and lethally. (Nature. 2005 Mar 31; 434 (7033): 598-604. Nature. 2005 Apr 14; 434 (7035): 917-21. Nature. 2005 Apr 14; 434 (7035): 913-7. Nature. 2005 Apr 14; 434 (7035): 864-70). Therefore, a method for screening a drug for BRCA1-amplified disease, comprising the step of evaluating one or more test compounds using BPA1-amplified disease as an index by reducing the activity of Parp protein or suppressing the expression of PARP gene. Provided. A method for screening a drug for a BRCA1-amplified disease, comprising supplying one or more test compounds to BRCA1- and / or BRCA2-amplified cells; and in the BRCA1- and / or BRCA2-amplified cells, There is also provided a screening method comprising the step of evaluating the one or more test compounds using, as an index, decreased activity of Parp protein or suppression of expression of a gene encoding the protein. In these screening methods, it is preferable to use BRCA1 and / or BRCA2 amplified cells, as in the screening method for MYCN-amplified diseases. Furthermore, it is preferable to use cells (including transformed cells) in which PARP is amplified in addition to BRCA1 and / or BRCA2.

  The condensin 1 complex is known to assist the action of the Parp protein (DNA repair). Therefore, the subunit protein of the condensin 1 complex and the gene encoding it can be molecular targets even in BRCA1-amplified diseases. Therefore, a compound that decreases (inhibits) the activity of the subunit protein or suppresses the expression of a gene encoding one or more of the proteins is a useful drug or candidate compound even in a BRCA1-amplified disease.

(Drug for MYCN amplification disease)
The drug for MYCN amplification type disease disclosed in the present specification is composed of Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein which are subunit proteins constituting the condensin 1 complex. A compound that reduces the activity of one or more proteins selected from the group or suppresses the expression of genes encoding these proteins can be used as an active ingredient. As described above, such a compound is a drug or candidate compound that selectively acts on MYCN-amplified cells by avoiding or suppressing the action on the MYCN gene in MYCN-amplified diseases.

  The compound is not particularly limited. In addition to low-molecular organic compounds that can be inhibitors of the above proteins, anti-RNAs and siRNAs designed to suppress the expression of the above-mentioned genes can be mentioned.

(Prognostic marker in MYCN amplification disease)
The prognostic marker in the MYCN-amplified disease disclosed in the present specification consists of Smc2 protein, Smc4 protein, CAP-D2 protein, CAP-H protein and CAP-G protein which are subunit proteins constituting the condensin 1 complex. An expression product of a gene encoding one or more proteins selected from the group can be included. The expression products of genes encoding these proteins, that is, mRNA and protein, increase in expression in the MYCN amplification type disease as the MYCN gene is amplified. Therefore, it is known that the prognosis is worse in the MYCN amplification type disease as the degree of enhancement of the gene is larger. Therefore, the expression products of these genes can be used as prognostic markers for MYCN-amplified diseases. In order to use the expression products of these genes as markers, for example, mRNAs that are transcription products of these genes and primers or probes that can qualitatively and / or quantitatively detect cDNA obtained from mRNA, or these An antibody for detecting a protein that is a translation product of a gene and a reagent for detecting the activity of the protein can be used. When the amino acid sequence of a protein used as a marker or the base sequence of a gene is known, those skilled in the art can appropriately obtain it based on a known technique. This prognostic marker can be preferably applied to neuroblastoma.

  The marker is preferably an expression product of a gene encoding one or more proteins of Smc2, Smc4, CAP-D2 and CAP-H, more preferably one of Smc2, Smc4 and CAP-D2. Or it is the expression product of the gene which codes 2 or more types of protein, More preferably, it is the expression product of the gene which codes Smc2 protein.

(Prognosis prediction method)
The prognostic method for predicting the prognosis of MYCN-amplified disease disclosed in the present specification is based on a test sample collected from a patient with MYCN-amplified disease. Smc2 protein, Smc4 protein, CAP, which are subunits constituting a condensin 1 complex Predicting the prognosis of the MYCN-amplified disease using as an index the expression status of a gene encoding one or more proteins selected from the group consisting of -D2 protein, CAP-H protein and CAP-G protein Can do. The expression status of genes encoding these proteins increases in accordance with the amplification status of the MYCN gene in the MYCN-amplified disease, that is, the degree of progression of the disease, and also corresponds to poor prognosis. Therefore, the prognosis (progression level) of MYCN-amplified disease can be predicted by using the expression status of these genes as an index.

  The expression of these genes is performed by qualitatively or quantitatively detecting the expression product of the gene encoding the above-described protein. For example, it can be carried out by detecting the amount of mRNA that is the transcription product of the gene, the protein that is the translation product of the gene and its activity, or measuring the expression level of the protein and the activity of the protein. As described above, these techniques can be appropriately implemented by those skilled in the art by applying known techniques.

  Hereinafter, specific examples embodying the disclosure of this specification will be shown. The disclosure of the present specification is not limited to the following examples.

  The experimental methods used in the examples are described below.

(1) Mouse
MYCN transgenic mice (neuroblastoma model mice) were prepared by the present inventor Kenji Kadomatsu and others, Graduate School of Medicine, Nagoya University (Postal Code 466-8550, Showa-ku, Nagoya, Aichi, Japan) At 65 Tsurumaicho), standard feed and water are supplied and maintained in a controlled environment and are available. Normal ganglia, precancerous and cancerous tissues were excised from wild-type mice, hemizygous and homozygous MYCN transgenic mice, ground, and total RNA was extracted according to a conventional method.

(2) Cell line
IMR32 cells (human neuroblastoma cells, multiple copies) were cultured in MEM medium (containing 10% fetal calf serum, 1% non-essential amino acids, penicillin-streptomycin). SK-N-AS (human neuroblastoma cells, single copy), SK-N-BE (same, multiple copies), SH-SY5Y (same, single copy) and 293T cells are Dulbecco's modified Eagle medium (10% fetal bovine Serum-containing). SH-EP cells (single copy) were cultured in RPMI 1640 medium (10% fetal bovine serum and penicillin-streptomycin).

(3) Virus infection Transfer the lentiviral packaging plasmid psPAX2, pMD2.G, non-target vector, shRNA expression vector (Openbiosystems) and expression vector (RIKEN) into 293T cells using FuGENE HD (Promega). Erected. The culture supernatant 3 days and 4 days after transfection was used as a virus solution.

(4) Plasmid
sh RNA non-target (Sigma), sh Smc2-38, 40 and 42 (Openbiosystems) were purchased.

(5) DNA damage reagent The cells were treated with cisplatin or CPT (camptothecin) to damage chromosomal DNA.

(6) RNA isolation, RT-PCR and quantitative real-time PCR
Total RNA was isolated from predetermined cells or tissues using ISOGEN II (Nippongene, Japan). For RT-PCR and quantitative real-time PCR, total RNA (1 μg) was incubated with DNase I (Invitrogen, Carlsbad, Calif.) To remove contaminating genomic DNA. Then, it used for RT-PCR with oligo dT, a random hexamer primer, and ThermoScript RT-PCR system. hSmc2, hGAPDH (control) and various amounts gene was determined by [Delta] [Delta] _T method (Mx3000p or Mx3005p (Agilent technologies) ).

(7) FACS
Samples for flow cytometry were fixed in 70% ethanol, washed with 1 ml PBS, resuspended in 1 ml PBS containing 100 μg / ml RNase A, and incubated at 37 ° C. for 1 hour. To this solution, iodopropidium was added so that the total amount was 50 μg / ml, and the mixture was incubated at 4 ° C. for 30 minutes. This solution was tested using a Becton-Dickinson FACS calibur according to the attached protocol.

(8) TUNEL assay
A TUNEL assay was performed using APO-DIRECT kit (556381, BD pharmingen). Cell fixation and staining were performed according to the protocol attached to the kit. Subsequently, the TUNEL assay was performed using FACScanto (556381, BD pharmingen) for the sample by APO-DIRECT.

(9) Fluorescent immunostaining For fluorescent immunostaining, cells were cultured on glass coverslips in 4 or 6 well plates until 50-70% confluent. The coverslip was washed twice with PBS and then fixed with 4% paraformaldehyde for 1 hour at room temperature. Subsequently, the cells were washed 3 times with PBS. Furthermore, after leaving still for 30 minutes at room temperature with 1% Triton / PBS, it was washed 3 times with PBS, allowed to stand for 30 minutes at room temperature with 2% BSA / PBS, and washed 3 times with PBS. This solution was reacted with the primary antibody (room temperature for 1 hour). The primary antibody (γ-H2AX antibody (Millipore), a marker for DNA damage) was diluted 1000 times with a blocking solution. The reaction solution was washed 3 times with PBS, a secondary antibody (mouse-Alexa488) (diluted 1000 times) was added, and the mixture was allowed to stand at room temperature for 30 minutes. The reaction solution was washed 3 times with PBS, stained with DAPI (4 ′, 6′-diamidino-2-phenylindole) for 10 minutes, and then observed under a microscope.

(Evaluation of Smc2 expression in mice)
The expression level of Smc2 in hyperplasia and tumor ganglion in wild type mice (2 weeks old) and MYCN transgenic mice (homozygotes and hemizygotes, 2 and 4 weeks of age) was evaluated. According to the above (1) and (6), total RNA was extracted from a predetermined mouse, and RT-PCR and real-time PCR were performed. The results are shown in FIGS.

  As shown in FIG. 3, the expression of Smc2 was weak in the wild type mouse, but in the MYCN transgenic mouse (neuroblastoma model mouse), as the age increased, ganglion changed from the hyperplastic cell to the tumor. It turned into a cell and it turned out that canceration is progressing. Moreover, it turned out that the expression level of Smc2 increases with canceration. Furthermore, as shown in FIG. 4, it was found that the expression of Smc2 already increased in the hyperplastic ganglion of the MYCN transgenic mouse (hemizygote, 2 weeks old).

  The expression level of Smc2 was evaluated using the human neuroblastoma cell line (MYCN gene multicopy and single copy) described in (2) above. The results are shown in FIGS.

  As shown in FIG. 5, in the cell line having a single copy of the MYCN gene (SH-SY5Y and SK-N-AS), almost no expression of Smc2 is observed, whereas the MYCN gene amplified cell line (IMR32 and SK- In N-BE), it was found that the expression of Smc2 was increased. It was also found that when MYCN gene was introduced into cells with normal MYCN gene and MYCN was forcibly expressed, increased expression of Smc2 was induced.

  The effect of Smc2 knockdown on human neuroblastoma cell lines (multicopy and single copy) was evaluated. A shRNA vector targeting Smc2 and a non-target vector serving as a control prepared according to (3) and (4) above to the IMR32 cells (human neuroblastoma cell line, multiple copies) described in (2) above. The virus solution containing each was infected by a conventional method. These cell lines were cultured and the number of cells was counted. The results are shown in FIG.

  As shown in FIG. 7, in the MYCN gene-amplified cell line (IMR32), cell growth was suppressed when Smc2 was knocked down, and when it was not knocked down, the cells grew. In contrast, in the MYCN gene single copy cell line (SK-N-AS), the cells proliferated whether or not Smc2 was knocked down. From these results, it was found that Smc2 is necessary for cell proliferation in the MYCN gene amplified cell line.

  It was confirmed whether suppression of cell proliferation by Smc2 knockdown in the MYCN gene amplified cell line (IMR32) was due to apoptosis. In Example 3, except that a virus solution containing the shsmc2-40 vector was prepared, a virus solution was prepared in the same manner as in Example 3 to infect the IMR32 cell line. The prepared Smc2 knockdown IMR32 cell line was subjected to a TUNEL assay according to (8) above. The results are shown in FIGS.

  As shown in FIG. 8, in the Smc2 knockdown IMR32 cell line into which the shsmc2-40 vector was introduced, it was found that 97.1% of the cells reached apoptosis. In contrast, in the uninfected IMR32 cell line and the IMR32 cell line infected with the control vector, almost no cells leading to apoptosis were observed. In addition, as shown in FIG. 9, it was found that in the Smc2 knockdown IMR32 cell line, cells leading to apoptosis increase as the culture progresses. From the above, it was found that one of the reasons for the suppression of cell proliferation by Smc2 knockdown in the MYCN gene amplified cell line was due to apoptosis. That is, Smc2 was found to be a synthetic lethal acting gene by suppressing its expression in an abnormal cell line in which the MYCN gene is amplified.

  The function of the synthetic lethal gene SMC2 in the MYCN gene amplified cell line was analyzed. In order to confirm DNA damage in the Smc2 knockdown IMR32 cell line and the Smc2 knockdown SK-N-AS cell line and the non-target IMR32 cell line and the non-target SK-N-AS cell line prepared in Example 3, the above (9 ) Was subjected to fluorescent immunostaining. The results are shown in FIG. 10, FIG. 11 and FIG.

  As shown in FIGS. 10 and 11, in the MYCN gene-amplified cell line, when Smc2 was knocked down, DNA damage increased compared to the case of not knocking down, whereas the MYCN gene single-copy cell line , DNA damage was similar regardless of the presence or absence of Smc2 knockdown. As shown in FIG. 12, which summarizes these results, it was found that the increase in DNA damage due to Smc2 knockdown was exceptional in the MYCN gene amplified cell line. From the above results, it was found that knockdown of Smc2 may induce MYCN-induced DNA damage repair failure.

  After DNA damage reagents (cisplatin and camptothecin) were supplied to the MYCN gene single copy cell line (SH-EP) to cause DNA damage, a virus solution containing the shSmc2 vector was prepared in the same manner as in Example 2. Infected according to a conventional method, apoptosis was observed according to (8) above. The results are shown in FIG.

  As shown in FIG. 13, it was found that apoptosis occurred when Smc2 was knocked down as compared to the non-target. From this result, it was strongly speculated that Smc2 is related to DNA repair, and that Smc2 knockdown induces DNA repair failure. FIG. 14 shows the ontology of molecular groups that correlate with the expression of Smc2 using neuroblastoma clinical specimens. As shown in FIG. 14, many molecules that correlated with Smc2 were related to DNA repair. This supports that DNA repair failure is induced by knockdown of Smc2.

  Expression analysis of the Smc2 knockdown MYCN gene amplified cell line (IMR32 cells) and the Smc2 knockdown MYCN gene single copy cell line (SK-N-AS cells) prepared in Example 2 was performed according to a conventional method. These results are shown in FIGS. 15 and 16, respectively.

  As shown in FIGS. 15 and 16, in the MYCN gene-amplified cells, the expression level of a group of molecules involved in DNA repair is remarkably reduced by knocking down Smc2, but in the MYCN gene single-copy cells, these molecules are reduced. No significant decrease was observed in the expression level of the group.

From the above results, the following can be understood. FIG. 17 shows the growth mechanism of MYCN gene amplified cells, and FIG. 18 shows the growth mechanism of MYCN gene single copy cells. Overexpression of MYCN causes cell proliferation but at the same time causes DNA damage, which normally leads to cell death. However, as shown in the left column of FIG. 17, MYCN amplification simultaneously induces Smc2 expression. For this reason, DNA repair is promoted, and infinite proliferation ability is acquired as cancer cells. On the other hand, as shown in the right column of FIG. 17, when Smc2 is knocked down, the cells die because of DNA repair failure.
As shown in the left column of FIG. 18, when the MYCN gene is a single copy cell, the role of Smc2 is small because the DNA damage is not so large, and the cell proliferates. For this reason, even if Smc2 is knocked down, the cells do not die (right column in FIG. 18).

  Based on the above results, data published on the web (NCBI GSE-3960) and corresponding prognostic data in patients with neuroblastoma (John M Maris group (Giulio D'Angio Chair in Neuroblastoma Research, Based on Chief, Division of Oncology, Director, Center for Childhood Cancer Research, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine)), the following analysis was performed. The analysis was requested by Associate Professor Ichiro Takeuchi, Department of Computer Science and Engineering, Nagoya Institute of Technology. The results are shown in FIGS.

  FIGS. 19 and 20 show the relationship between Smc2 expression in MYCN amplified and non-MYCN amplified patients (but expressing Smc2) in neuroblastoma patients. As shown in FIGS. 19 and 20, Smc2 expression was associated with prognosis only in MYCN-amplified patients.

  FIG. 21 shows a subunit protein and another condensin 2 complex of a condensin 1 complex containing Smc2 as a subunit for a MYCN amplified patient and a non-MYCN amplified patient (but expressing Smc2) in a neuroblastoma patient. The expression profile of subunit protein is shown. As shown in the upper part of FIG. 21, it was found that there was a strong correlation between the expression or amplification of MYCN and the gene group constituting the condensin 1 complex. On the other hand, it was found that the gene group constituting the condensin 2 complex had no correlation with the expression and amplification of Smc2 (lower part of FIG. 21).

  FIG. 22 shows subunit proteins constituting a condensin 1 complex in MYCN-amplified patients and non-MYCN-amplified patients (however, expressing Smc2) in neuroblastoma patients, and hcapD2, hCAP-H other than Smc2 and It is a table | surface which shows the p value of the expression level and prognosis of hCAP-D3 and hCAP-H2 which comprise a condensin 2 complex, and Smc4 common to these complexes. As shown in FIG. 22, it was found that there was a strong correlation with the expression levels of hCAP-D2, hCAP-H and Smc4 in MYCN amplification type patients.

  From the above, molecular targets (compounds or genes, in the present case, for screening effective drugs for such diseases by searching for genes that act synthetically and lethally in diseases associated with genetic abnormalities) It was found that a subunit protein of a condensin 1 complex or a gene encoding these proteins can be found.

  It was also found that drugs for diseases associated with genetic abnormalities can be screened by searching for compounds that act on molecular targets thus obtained.

Furthermore, it was found that the expression level of the constituent subunit protein of condensin complex 1 can be a prognostic marker for neuroblastoma patients with MYCN gene amplification. Since the condensin 1 complex functions as an assembly of these subunits, these subunit proteins or genes encoding them are used as molecular targets to reduce their activity or suppress their expression. It has been found that the compound can be a useful drug for MYCN-amplified neuroblastoma.

Claims (7)

A method for screening a drug for MYCN amplified disease,
A screening method comprising a step of evaluating one or more test compounds using as an index the suppression of expression of a gene encoding a Smc2 protein that is a subunit protein constituting a condensin 1 complex . The screening method according to claim 1 , wherein the MYCN amplification type disease includes neuroblastoma. A method for screening a drug for MYCN amplified disease,
Supplying one or more test compounds to MYCN-amplified cells;
Evaluating the one or more test compounds using as an index the suppression of expression of a gene encoding the Smc2 protein, which is a subunit protein constituting the condensin 1 complex, in the MYCN-amplified cell ;
A screening method comprising: The screening method according to claim 3 , wherein the MYCN amplification disease includes neuroblastoma. A drug for MYCN-amplified diseases comprising, as an active ingredient, an shRNA expression vector that suppresses the expression of a gene encoding Smc2 protein that is a subunit protein constituting the condensin 1 complex . A method for predicting the prognosis of a MYCN-amplified disease for a test sample collected from a patient with an MYCN-amplified disease, using the expression status of a gene encoding the Smc2 protein constituting the condensin 1 complex as an index . The method according to claim 6 , wherein the MYCN-amplified disease includes neuroblastoma.