WO2015125956A1 - p300を阻害する化合物によるがんの治療への応答性を予測する方法 - Google Patents
p300を阻害する化合物によるがんの治療への応答性を予測する方法 Download PDFInfo
- Publication number
- WO2015125956A1 WO2015125956A1 PCT/JP2015/054991 JP2015054991W WO2015125956A1 WO 2015125956 A1 WO2015125956 A1 WO 2015125956A1 JP 2015054991 W JP2015054991 W JP 2015054991W WO 2015125956 A1 WO2015125956 A1 WO 2015125956A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cbp
- cancer
- compound
- cells
- dna
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/415—1,2-Diazoles
- A61K31/4155—1,2-Diazoles non condensed and containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91045—Acyltransferases (2.3)
- G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
- G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to a method for predicting responsiveness to cancer treatment by a compound that inhibits p300, using CBP function suppression as an index, and a method for selecting cancer treatment targets.
- the present invention also relates to a method for treating cancer in which CBP function suppression is caused by a compound that inhibits p300.
- this invention relates to the reagent for detecting the presence or absence of the function suppression of CBP used for these methods.
- the present invention relates to a method for screening a compound for use in the treatment of cancer in which CBP function suppression has occurred using p300 inhibition as an index.
- the tyrosine kinase inhibitor is effective for solid cancers accompanied by activating mutations in tyrosine kinase genes such as EGFR mutation and ALK fusion found in lung adenocarcinoma (Non-patent Document 1).
- tyrosine kinase genes such as EGFR mutation and ALK fusion found in lung adenocarcinoma
- Non-patent Document 2 We and other researchers recently identified a RET oncogene fusion in lung adenocarcinoma, which supports the importance of tyrosine kinase genes as therapeutic targets.
- chromatin regulatory proteins eg, histone acetyltransferases CBP / CREBBP and p300 / EP300, histone methyltransferases MLL2 and SETD2, histone demethylases JARID1C and UTX, chromatin remodeling factor BRG1, Inactivated somatic mutations in genes encoding ARID1A, ARID2, PBRM1, and SNF5 have been of great interest since they were first identified by genome-wide sequence analysis of cancer cells. These mutations are thought to inhibit functions in transcription and DNA double-strand break repair, and appear to have critical implications for cancer development and / or progression.
- chromatin regulatory proteins eg, histone acetyltransferases CBP / CREBBP and p300 / EP300, histone methyltransferases MLL2 and SETD2, histone demethylases JARID1C and UTX, chromatin remodeling factor BRG1, Inactivated somatic mutations in genes encoding ARID1A, ARID
- the present invention has been made in view of such circumstances, and an object thereof is to develop a therapeutic strategy for specifically targeting cancer cells having CBP function suppression.
- Synthetic lethal therapy is a very promising cancer treatment.
- the BRCA1 and BRCA2 genes have a synthetic lethal relationship with the PARP1 gene.
- the growth of BRCA1-deficient or BRCA2-deficient cancer cells depends on the function of the PARP1 protein. And this finding has shifted to clinical practice in the form of the development of PARP inhibitors for treating BRCA1 / BRCA2 deficient tumors (Chan DA, et al. Nat Rev Drug Discov. 2011; 10: 351- 64).
- synthetic lethal therapy has been proposed in the treatment of cancers deficient in genes involved in DNA mismatch repair and cell metabolism (Muller FL, et al. Nature. 2012; 488: 337-42, Chan DA. , Et al.
- the present inventors have intensively studied to apply synthetic lethal therapy to a therapeutic strategy for specifically killing cancer cells having a CBP mutation.
- the present inventors significantly suppressed the growth of the cancer cells when the expression suppression or function inhibition of the p300 protein was performed in cancer cells having a CBP mutation.
- the inventors have found that such growth inhibition does not occur.
- the ratio of positive cells was increased by Annexin V / PI staining, so it was found that apoptosis was induced.
- the growth inhibitory effect of cancer cells having a CBP mutation by suppressing the expression of p300 was also demonstrated in vivo.
- the present inventors have found that screening for drugs useful for the treatment of CBP mutant cancer can be performed using whether or not p300 is inhibited as an index, and the present invention has been completed.
- the present invention relates to a synthetic lethal therapy for specifically targeting cancer cells having CBP function suppression such as CBP mutation and a companion diagnosis for the synthetic lethal therapy.
- This invention is provided.
- a compound that uses a biological sample derived from a cancer patient detects the presence or absence of functional suppression of CBP contained in the biological sample, and inhibits p300 from a patient in which functional suppression of CBP is detected
- a method for predicting responsiveness to a cancer treatment by a compound that inhibits p300 comprising determining that the compound is responsive to the treatment of cancer.
- a method for selecting a target for cancer treatment with a compound that inhibits p300 comprising selecting as a target for cancer treatment.
- Oligonucleotide primer that specifically binds to CBP gene
- Oligonucleotide probe that specifically binds to CBP gene
- Antibody that specifically binds to CBP protein
- Inhibition of CBP function A method for screening a compound for use in the treatment of cancer, comprising a step of selecting a compound using whether p300 is inhibited or not as an index.
- a therapeutic agent for cancer comprising a compound that inhibits p300, wherein CBP function suppression is detected.
- the present invention it became possible to efficiently predict the responsiveness to cancer treatment by a p300 inhibitor, using CBP function suppression as an index.
- CBP function suppression for example, inactivating mutation or decreased expression
- the patient can be treated for cancer with a p300 inhibitor. For this reason, it becomes possible to greatly improve the treatment results of cancer patients.
- a probe or primer for the CBP gene and an antibody against CBP it becomes possible to efficiently perform a companion diagnosis by detecting the presence or absence of such CBP function suppression.
- A is a photograph showing the results of Western blotting
- B is a graph showing cell viability
- C is a graph showing colony formation rate.
- It is a figure which shows the effect of expression suppression of p300 in a normal cell line.
- the top is a graph showing the results of detecting cell proliferation in each normal cell line
- the bottom is a photograph confirming that p300 disappearance is caused by siRNA in each normal cell line.
- A is a graph showing the survival rate of lung cancer cell lines
- B is a graph showing the IC50 of C646 against lung cancer cell lines
- C is a graph showing the survival rate of lymphoma cell lines
- D is a graph showing the IC50 of C646 against lymphoma cell lines. It is a figure which shows the mechanism of the cell death by the expression suppression of p300 in the cancer cell line which has a CBP mutation.
- the top is a diagram showing a general relationship between cell death and apoptosis, cell aging, and autophagy, and the bottom is a photograph in which changes of various markers are detected.
- the top is a diagram showing the outline of the experiment, and the bottom is the measurement of tumor growth in mice transplanted with control cancer cells (left) and cancer cells in which p300 expression is suppressed by the action of Dox (right). It is a graph which shows the result. It is a figure which shows the variation
- “HoD” indicates a homo-deletion and “HeD” indicates a hetero-deletion.
- N represents a nonsense mutation
- “F” represents a frameshift mutation
- “MD” represents a missense mutation in the domain
- M” represents a missense mutation.
- HAT histone acetyltransferase
- tumor ⁇ Methods for predicting responsiveness to cancer treatment, methods for selecting cancer treatment targets>
- tumor malignant tumor, cancer, malignant neoplasm, carcinoma, sarcoma, etc.
- cancer malignant neoplasm, carcinoma, sarcoma, etc.
- CBP and p300 have a synthetic lethal relationship in cancer cells, and in cancer cells in which CBP function suppression occurs, inhibition of p300 can suppress the growth of the cancer cells. It was issued. Based on this finding, responsiveness to cancer treatment by a compound that inhibits p300 can be evaluated using CBP function suppression as an index.
- the present invention uses a biological sample derived from a cancer patient, detects the presence or absence of functional suppression of CBP contained in the biological sample, and designates a patient whose functional suppression of CBP is detected as p300.
- the patient in which suppression of CBP function is detected in this manner is suitable for the treatment of cancer with a compound that inhibits p300, and the treatment of cancer with a compound that inhibits p300 is effective using the suppression of CBP function as an index. It is possible to select patients and ineffective patients and perform efficient treatment. Accordingly, the present invention uses a biological sample derived from a cancer patient, detects the presence or absence of CBP function suppression in the biological sample, and treats a patient whose CBP function suppression is detected as a compound that inhibits p300. There is also provided a method of selecting a target for cancer treatment with a compound that inhibits p300, which comprises selecting as a target for cancer treatment by.
- the “cancer patient” may be not only a human suffering from cancer but also a human suspected of suffering from cancer.
- the cancer patient that is the target for detecting the presence or absence of CBP function suppression, and all cancer patients can be targeted.
- the cancer in which CBP function suppression is observed may be, for example, lung cancer, bladder cancer, lymphoma, adenoid cyst cancer, and the like. Since CBP is known to be mutated in a subset of 10% of lung cancer, 13% of bladder cancer, 20-40% of lymphoma, and 7% of adenoid cystic cancer, the method of the present invention Therefore, cancer patients with such a frequency can be selected as “targets of treatment”.
- the “biological sample” used in the present invention is not particularly limited as long as it can detect the presence or absence of CBP function suppression, but is preferably a cancer biopsy specimen. It may be a protein extract or a nucleic acid extract obtained from these samples (mRNA extract, cDNA preparation or cRNA preparation prepared from the mRNA extract, etc.).
- CBP and “p300” in the present invention are both histone acetyltransferases involved in chromatin control, and both are paralogous.
- a typical base sequence of human-derived natural CBP genomic DNA is shown in SEQ ID NO: 1
- a typical base sequence of human-derived natural CBP cDNA is shown in SEQ ID NO: 2
- a typical human-derived natural CBP protein is typical.
- a typical amino acid sequence is shown in SEQ ID NO: 3.
- a typical base sequence of human-derived natural p300 genomic DNA is shown in SEQ ID NO: 4
- a typical base sequence of human-derived natural p300 cDNA is shown in SEQ ID NO: 5
- a human-derived natural p300 protein is shown in SEQ ID NO: 6. It should be understood that individual differences may occur in the sequence due to polymorphism and the like even in CBP and p300 that have not undergone mutation.
- “Inhibition of CBP function” in the present invention includes both CBP inactivation, activity reduction and expression reduction.
- CBP inactivation is typically due to inactive mutations in CBP.
- Inactive mutations include, for example, missense mutations in the histone acetyltransferase (HAT) domain of CBP (positions 1342-1648 of the amino acid sequence described in SEQ ID NO: 3), nonsense mutations over the entire region, whole genes or Although it may be caused by partial deletion or the like, it is not limited to these as long as CBP is inactivated. Examples of CBP mutations are shown in Table 1 and FIG. Abbreviations in Table 1 are as follows.
- SCC small cell carcinoma
- AdC adenocarcinoma
- SqC squamous cell carcinoma
- LCC large cell carcinoma
- NT untested
- ND undetected. * Abnormal size.
- Corresponding non-cancerous tissue DNA is not available.
- the reduction in the expression of CBP includes both the reduction in the expression at the transcription level and the reduction in the expression at the translation level.
- the method of “detection of CBP function suppression” in the present invention is not particularly limited, and examples thereof include the following methods.
- detecting a mutation means, in principle, detecting a mutation in the genomic DNA, but the mutation in the genomic DNA causes a change in the base in the transcript or an amino acid in the translation product. In the case of reflection, it means that the change in the transcript or translation product is detected (that is, indirect detection).
- a preferred embodiment of the method of the present invention is a method for detecting a mutation by directly determining the base sequence of the CBP gene region of a cancer patient.
- the “CBP gene region” means a certain region on the genomic DNA containing the CBP gene.
- the region includes an expression control region (for example, a promoter region and an enhancer region) of the CBP gene and a 3 'terminal untranslated region of the CBP gene. Mutations in these regions can affect, for example, the transcriptional activity of the CBP gene.
- a DNA sample is first prepared from a biological sample derived from a cancer patient.
- DNA samples include genomic DNA samples and cDNA samples prepared by reverse transcription from RNA.
- a method for extracting genomic DNA or RNA from a biological sample is not particularly limited, and a known method can be appropriately selected and used.
- a method for extracting genomic DNA an SDS phenol method (urea is used).
- a method in which a tissue stored in ethanol or a solution containing the protein is denatured with proteinase (proteinase K), surfactant (SDS), and phenol, and the protein in the tissue is denatured and precipitated from the tissue with ethanol.
- RNA extraction method using phenol and chaotropic salt for example, an extraction method using phenol and chaotropic salt (more specifically, commercially available kits such as Trizol (manufactured by Invitrogen), Isogen (manufactured by Wako Pure Chemical Industries, Ltd.), etc. are used.
- RNAPrep total RNA extraction kit manufactured by Beckman Coulter
- RNeasy Mini manufactured by QIAGEN
- RNA Extraction Kit manufactured by Pharmacia Biotech
- the reverse transcriptase used for preparing cDNA from the extracted RNA is not particularly limited, and for example, a reverse transcriptase derived from a retrovirus such as RAV (Rouse associated virus) or AMV (Avian myeloblastosis virus).
- a reverse transcriptase derived from a retrovirus of a mouse such as MMLV (Moloney murine leukemia virus).
- DNA containing a mutation site in the CBP gene region is isolated, and the base sequence of the isolated DNA is determined.
- the DNA can be isolated, for example, by PCR or the like using genomic DNA or RNA as a template, using a pair of oligonucleotide primers designed to sandwich a mutation in the CBP gene region.
- the base sequence of the isolated DNA can be determined by methods known to those skilled in the art, such as the Maxam Gilbert method and the Sanger method.
- the presence or absence of mutations in the CBP gene region in cancer cells of cancer patients by comparing the determined DNA or cDNA nucleotide sequences with controls (eg, DNA or cDNA nucleotide sequences derived from non-cancerous tissue of the same patient) Can be determined.
- controls eg, DNA or cDNA nucleotide sequences derived from non-cancerous tissue of the same patient
- the method for detecting a mutation in the CBP gene region can be performed by various methods capable of detecting a mutation other than the method for directly determining the base sequence of DNA or cDNA.
- the detection of the mutation in the present invention can also be performed by the following method.
- a DNA or cDNA sample is prepared from a biological sample.
- an oligonucleotide probe having a base sequence complementary to the base sequence containing a mutation in the CBP gene region and labeled with a reporter fluorescent dye and a quencher fluorescent dye is prepared.
- the oligonucleotide probe is hybridized to the DNA sample, and the base sequence containing the mutation in the CBP gene region is amplified using the DNA sample hybridized with the oligonucleotide probe as a template.
- the fluorescence emitted from the reporter fluorescent dye is detected by the degradation of the oligonucleotide probe accompanying the amplification, and then the detected fluorescence is compared with a control.
- a method include a double die probe method, a so-called TaqMan (registered trademark) probe method.
- a DNA or cDNA sample is prepared from a biological sample.
- a reaction system including an intercalator that emits fluorescence when inserted between DNA double strands a base sequence containing the mutation in the CBP gene region is amplified using the DNA sample as a template.
- the temperature of the reaction system is changed, a change in the intensity of the fluorescence emitted by the intercalator is detected, and the change in the intensity of the fluorescence accompanying the detected change in the temperature is compared with a control.
- An example of such a method is an HRM (high resolution melting, high resolution melting curve analysis) method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- the amplified DNA is cleaved with a restriction enzyme.
- the DNA fragments are separated according to their sizes. The size of the detected DNA fragment is then compared to a control.
- Examples of such a method include a method using a restriction enzyme fragment length mutation (Restriction Fragment Length Polymorphism / RFLP) and a PCR-RFLP method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- the amplified DNA is dissociated into single-stranded DNA.
- the dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with the control.
- An example of such a method is a PCR-SSCP (single-strand conformation polymorphism, single-strand conformation mutation) method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- the amplified DNA is separated on a gel where the concentration of the DNA denaturant is gradually increased. The mobility of the separated DNA on the gel is then compared to the control.
- An example of such a method is a denaturant gradient gel electrophoresis (DGGE) method.
- DGGE denaturant gradient gel electrophoresis
- a method using DNA containing a mutation site in the CBP gene region prepared from a biological sample, and a substrate on which an oligonucleotide probe that hybridizes to the DNA is immobilized examples include a DNA array method.
- a DNA or cDNA sample is first prepared from a biological sample.
- an oligonucleotide primer having a base sequence complementary to the base sequence on the 1 ′ base 3 ′ side and the base sequence on the 3 ′ side of the base of the mutation site in the CBP gene region is prepared.
- a ddNTP primer extension reaction is performed using the DNA as a template and the primer.
- the primer extension reaction product is applied to a mass spectrometer, and mass measurement is performed.
- the genotype is determined from the mass measurement result. The determined genotype is then compared to a control.
- An example of such a method is the MALDI-TOF / MS method.
- a DNA or cDNA sample is first prepared from a biological sample.
- 5 ′-“base of mutation site in CBP gene region and base sequence complementary to 5′-side base sequence” “one base 3 ′ side of mutation site in CBP gene region and 3′-side thereof
- An oligonucleotide probe consisting of a base sequence that does not hybridize to the base sequence "-3 '(flap) is prepared.
- an oligonucleotide probe having a base sequence complementary to the base of the mutation site in the CBP gene region and its 3′-side base sequence” is prepared.
- the two kinds of oligonucleotide probes are hybridized to the prepared DNA.
- the hybridized DNA is cleaved with a single-stranded DNA cleaving enzyme to release the flap.
- a single-strand DNA cutting enzyme For example, cleavase is mentioned.
- an oligonucleotide probe having a sequence complementary to the flap and labeled with reporter fluorescence and quencher fluorescence is then hybridized to the flap.
- the intensity of the generated fluorescence is measured.
- the measured fluorescence intensity is then compared to a control.
- An example of such a method is the Invader method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- the amplified DNA is dissociated into single strands, and only one strand is separated from the dissociated single strand DNA.
- an extension reaction is performed one by one from the vicinity of the base at the mutation site in the CBP gene region.
- the pyrophosphate produced at that time is enzymatically luminescent, and the intensity of luminescence is measured.
- the measured fluorescence intensity is then compared with the control.
- An example of such a method is a pyrosequencing method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- an oligonucleotide primer having a base on the 1 ′ base 3 ′ side of the base of the mutation site in the CBP gene region and a base sequence complementary to the 3 ′ side base sequence is prepared.
- a single base extension reaction is performed using the prepared DNA as a template and the prepared primer.
- the degree of polarization of fluorescence is measured. The measured degree of fluorescence polarization is then compared to the control.
- An example of such a method is the AcycloPrime method.
- a DNA or cDNA sample is first prepared from a biological sample.
- DNA containing a mutation site in the CBP gene region is amplified.
- an oligonucleotide primer having a base on the 1 ′ base 3 ′ side of the base of the mutation site in the CBP gene region and a base sequence complementary to the base sequence on the 3 ′ side is prepared.
- a single base extension reaction is performed using the amplified DNA as a template and the prepared primer.
- the base type used in the single base extension reaction is determined. The determined base species is then compared to a control.
- An example of such a method is the SNuPE method.
- the sample prepared from the biological sample may be a protein as long as the mutation is accompanied by an amino acid change (for example, substitution, deletion, insertion) in the CBP protein.
- an amino acid change for example, substitution, deletion, insertion
- a method using a molecule for example, an antibody
- a method for detecting a protein using an antibody will be described later.
- the “decreased expression of CBP” in the present invention usually means that the expression level is lower than that in a control (for example, the expression level in a non-cancerous tissue of a healthy person or the same patient).
- RNA or cDNA is prepared from a biological sample derived from a cancer patient by the above method. Then, an oligonucleotide primer or an oligonucleotide probe is used for an amplification reaction or a hybridization reaction, respectively, and the amplification product or hybrid product is detected.
- a method for example, RT-PCR method, Northern blot method, dot blot method, DNA array method, in situ hybridization method, RNase protection assay method, mRNA-seq and the like can be used.
- a person skilled in the art can design oligonucleotide primers and oligonucleotide probes suitable for each method based on the base sequence of CBP cDNA (for example, SEQ ID NO: 2) by conventional methods.
- a protein sample is prepared from a biological sample derived from a cancer patient.
- an antibody specific for the CBP protein is used for the antigen-antibody reaction, and the binding of the antibody to the CBP protein is detected.
- the CBP protein can be directly detected.
- a labeled molecule that recognizes the antibody for example, A CBP protein can be detected indirectly by using a secondary antibody or protein A) and utilizing the label of the molecule.
- Immunohistochemistry immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, analysis using antibody arrays, etc. can do.
- Immunohistochemistry also has the advantage that additional information such as the morphology and distribution of cancer cells in the tissue can be obtained at the same time.
- the type and origin of the antibody to be used is not particularly limited, but is preferably a monoclonal antibody.
- oligoclonal antibodies mixture of several to several tens of antibodies
- polyclonal antibodies can also be used.
- functional fragments of antibodies such as Fab, Fab ′, F (ab ′) 2, Fv, scFv, sc (Fv) 2, dsFv, and diabody, and multimers thereof (for example, dimers, trimers, tetramers, Polymer) can also be used.
- a commercially available product may be used as the anti-CBP antibody.
- CBP protein can also be detected using mass spectrometry (MS).
- MS mass spectrometry
- analysis by a mass spectrometer (LC / MS) connected to liquid chromatography is advantageous because it is sensitive.
- Measurement by mass spectrometry is, for example, preparing a protein from a biological sample, labeling the protein, fractionating the protein, subjecting the fractionated protein to mass spectrometry, and identifying the CBP protein from the mass spectrometry value. Can be done.
- an isotope labeling reagent known in the art can be used, and an appropriate labeling reagent can be obtained as a commercial product. Fractionation can also be performed by a method known in the art, for example, using a commercially available strong cation column or the like.
- the decrease in gene expression is caused by promoter hypermethylation. Therefore, in the detection of the presence or absence of CBP function suppression, it may be possible to detect the CBP gene promoter methylation as an index.
- a method of directly detecting a change in a base sequence after bisulfite treatment having an activity of converting methylated cytosine to uracil by base sequencing, or before bisulfite treatment A known method such as a method of detecting indirectly using a restriction endonuclease that can recognize (can cleave) but cannot recognize (cannot cleave) the base sequence after bisulfite treatment can be used.
- the patient can be determined to be responsive to cancer treatment with a compound that inhibits p300, and the patient is inhibited from p300.
- responsiveness to cancer treatment is an index indicating whether or not a compound that inhibits p300 can exert a therapeutic effect on cancer.
- a patient to be treated may be selected at a level at which moderate responsiveness can be expected.
- the patient when CBP function suppression is not observed, the patient can be excluded from cancer treatment with a compound that inhibits p300. Thereby, the success rate of treatment can be improved.
- the present invention uses a biological sample derived from a cancer patient, detects the presence or absence of functional suppression of CBP contained in the biological sample, and provides p300 for patients in whom functional suppression of CBP is detected.
- the “compound inhibiting p300” used in this treatment is not particularly limited, and may be a known compound or a compound identified by screening described below.
- Compounds that inhibit p300 are administered orally or parenterally to cancer patients in various forms such as tablets, powders, granules, capsules, solutions, etc., depending on their properties (for example, Intravenous administration, arterial administration, local administration).
- the dose is not particularly limited as long as it is an amount effective to inhibit p300 and treat cancer. What is necessary is just to select suitably according to the age of a cancer patient, a body weight, a symptom, a health condition, the progress of cancer, etc. other than the property of a compound.
- administration frequency According to the objective, it can select suitably, For example, the dosage per day may be administered once per day, or it divides into multiple times. May be.
- the dosage range is from about 0.01 mg / kg body weight to about 500 mg / kg body weight, preferably from about 0.1 mg / kg body weight to about 100 mg / kg per day. It is weight.
- it is preferably administered once a day or divided into 2 to 4 times and repeated at appropriate intervals.
- the daily amount may exceed the above amount depending on the judgment of the doctor.
- cancer to be treated examples include lung cancer, bladder cancer, lymphoma, adenoid cystic cancer and the like, but are not limited to these cancers.
- the present invention also provides a reagent for detecting the presence or absence of CBP function suppression in the above method, comprising a reagent having any of the following molecules (a) to (c) as an active ingredient: provide.
- Oligonucleotide primer that specifically binds to CBP gene
- Oligonucleotide probe that specifically binds to CBP gene
- Antibody that specifically binds to CBP protein
- the primer should be adapted to the above-mentioned method and the region to be amplified, and the amplification products of genes other than the CBP gene should not be generated as much as possible.
- the length of the oligonucleotide primer is usually 15 to 50 bases, preferably 15 to 30 bases, but may be longer depending on the method and purpose.
- the above-mentioned polynucleotide probe is used as a primer in accordance with the above-described method and the region to be hybridized, and other than the CBP gene. What is necessary is just to design so that the hybridization to the gene may not occur as much as possible.
- Those skilled in the art can design such an oligonucleotide probe by a conventional method.
- the length of the oligonucleotide probe is usually 15 to 200 bases, preferably 15 to 100 bases, more preferably 15 to 50 bases, but may be longer depending on the technique and purpose.
- Oligonucleotide probes are preferably used after being appropriately labeled.
- a labeling method a method of labeling by phosphorylating the 5 ′ end of the oligonucleotide with 32 P using T4 polynucleotide kinase, and a random hexamer oligonucleotide or the like using a DNA polymerase such as Klenow enzyme
- the primer include a method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin (random prime method or the like).
- the oligonucleotide primer and oligonucleotide probe of the present invention can be prepared by, for example, a commercially available oligonucleotide synthesizer.
- An oligonucleotide probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.
- the oligonucleotide primer and oligonucleotide probe of the present invention may not be composed only of natural nucleotides (deoxyribonucleotide (DNA) or ribonucleotide (RNA)), or a part of non-natural nucleotides or All may be configured.
- Non-natural nucleotides include PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid), ENA (registered trademark, 2′-O, 4′-C-Ethylene-bridged nucleic acid), and these. A complex is mentioned.
- the antibody that specifically binds to the CBP protein is a polyclonal antibody
- an immunized animal is immunized with an antigen (CBP protein, a partial peptide thereof, or a cell that expresses these), and the antiserum is used for conventional means.
- an antigen CBP protein, a partial peptide thereof, or a cell that expresses these
- the antiserum is used for conventional means.
- salting out, centrifugation, dialysis, column chromatography, etc. can be purified and obtained.
- Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.
- the hybridoma method typically includes the method of Kohler and Milstein (Kohler & Milstein, Nature 1975; 256: 495).
- the antibody-producing cells used in the cell fusion step in this method are animals immunized with antigens (such as CBP protein, partial peptides thereof, or cells expressing these) (for example, mice, rats, hamsters, rabbits, monkeys, Goat) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium. As the myeloma cells, various known cell lines can be used.
- the antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species.
- the hybridoma is produced, for example, by cell fusion between a spleen cell obtained from a mouse immunized with an antigen and a mouse myeloma cell, and a hybridoma that produces a monoclonal antibody specific for the CBP protein is obtained by subsequent screening. Obtainable.
- Monoclonal antibodies against CBP protein can be obtained by culturing hybridomas or from ascites of mammals to which hybridomas have been administered.
- DNA encoding the above antibody is cloned from a hybridoma or B cell and incorporated into an appropriate vector, which is then introduced into a host cell (eg, mammalian cell line, E. coli, yeast cell, insect cell, plant cell, etc.). ) And the production of the antibody of the present invention as a recombinant antibody (for example, PJ Delves, Antibody Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Mono- TI NDB). PRESS, Vandam AM, et al. Eur. J. Biochem. 1990; 192: 767-775).
- the DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector to transform the host cell.
- Host cells may be transformed by incorporating into expression vectors (see WO94 / 11523).
- the antibody can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium.
- the methods used in the usual purification of polypeptides can be used. If transgenic animals (such as cows, goats, sheep or pigs) in which an antibody gene is incorporated are produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.
- the obtained anti-CBP antibody When detecting the amount of antibody directly bound to CBP protein, the obtained anti-CBP antibody is directly labeled with an enzyme, a radioisotope, a fluorescent dye, an avidin-biotin system, or the like.
- the obtained anti-CBP antibody (primary antibody) does not need to be labeled.
- a labeled molecule that recognizes the antibody for example, a secondary antibody or protein A may be used.
- reagent of the present invention in addition to the above-mentioned molecule as an active ingredient, other components acceptable as a reagent such as sterilized water, physiological saline, buffering agent, preservative and the like can be included as necessary.
- the present invention also provides a method for screening a compound for use in the treatment of cancer in which CBP function suppression has occurred, the method comprising a step of selecting a compound using whether p300 is inhibited or not as an index.
- test compound applied to the screening system of the present invention is not particularly limited.
- the test compound may be a known derivative of a p300 inhibitor (for example, a derivative of C646).
- “inhibition of p300” includes both inhibition of p300 activity and inhibition of expression. Since it is considered that the disappearance of histone acetyltransferase activity of p300 contributes to the lethality of CBP and p300, the inhibition of p300 as a screening index is preferably the inhibition of histone acetyltransferase activity of p300. .
- a radioisotope detection method Liau OD, et al. J. Biol. Chem.
- a test compound In screening, a test compound is allowed to act on this detection system to detect subsequent histone acetyltransferase activity. As a result of the detection, if the activity is reduced as compared to the histone acetyltransferase activity of p300 in the control (for example, when no test compound is added), it can be evaluated that the activity of p300 is inhibited.
- the compound obtained by screening is preferably specific to p300 from the viewpoint of reducing side effects and the like. Whether or not p300 is specific can be evaluated, for example, by conducting a binding experiment or activity inhibition experiment for each molecule. Therefore, the screening of the present invention may include a step of selecting a compound using as an index whether or not p300 is specific.
- a test compound is allowed to act on cells expressing p300, and the subsequent expression of p300 may be detected at the transcription level or translation level by the method described above.
- a reporter assay system using an expression construct in which a reporter gene is linked downstream of the p300 promoter may be used.
- expression is decreased as compared to expression of p300 in a control (for example, when no test compound is added) (expression of a reporter that substitutes for it in a reporter system), expression of p300 is reduced. It can be evaluated as being inhibited.
- the compound identified by the screening of the present invention can be made into a pharmaceutical product by mixing with a pharmacologically acceptable carrier and formulating it by a known pharmaceutical method.
- pharmacologically acceptable carriers include sterilized water, physiological saline, vegetable oil, solvents, bases, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, fragrances, excipients, vehicles. , Preservatives, binders, diluents, tonicity agents, soothing agents, extenders, disintegrating agents, buffering agents, coating agents, lubricants, coloring agents, sweeteners, thickeners, flavoring agents, dissolution Auxiliary agents or other additives may be mentioned, but are not limited thereto.
- Example 1 Development of treatment strategy based on synthetic lethality of cancer with CBP mutation
- Cell lines A549, H1299, H157, SQ5, H1703, LK2, and H520 (NSCLC), RL, Lucy, RC-K8, U2932, Ramos, Farage, SUP-T1, WSU-NHL, VAL, SUDHL5, Jurkat, TE8, TE10 were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS).
- MRC-5 cells normal fibroblasts
- HEK293T cells immortalized renal epithelial cells
- RPE-1 cells immortalized retinal epithelial cells
- RNA ON-TARGET plus SMARTpool siRNA was used for knocking down various proteins.
- Lipofectamine RNAiMAX (Invitrogen) was used for transfection.
- Non-targeted siRNA (L-001810-10) was used as a negative control.
- SiRNAs 50 nM were transfected into cancer cell lines using Lipofectamine RNAiMAX (Invitrogen). Two days later, the cells were trypsinized, counted, replated at a specific number in a 6-well culture dish, and further cultured for 12 days (14 days for various HAT knockdowns) to form colonies. The cells were then fixed in a solution containing 50% (v / v) methanol / 0.01% (w / v) crystal violet for 5 minutes and the number of colonies counted.
- Viability assessment was determined by measuring intracellular ATP levels using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega). SiRNAs (50 nM) were transfected into cancer cell lines using Lipofectamine RNAiMAX (Invitrogen). Two days later, the cells were trypsinized, counted and replated at a specific number in a 96-well plate. To measure cell viability, CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) was added to the cells, and fluorescence was measured with Envision (PerkinElmer).
- Annexin V-FITC / PI apoptosis detection kit (Roche) was used according to the manufacturer's instructions. That is, the cell pellet was suspended in 1 ⁇ binding buffer and incubated with Annexin V-FITC and PI for 20 minutes in the dark. Cell fluorescence was then analyzed by flow cytometry.
- shRNA Lentivirus In order to produce a tet-inducible cell line, pTRIPZ (Open Biosystems) derived from a shRNA expression lentiviral vector was transduced into the cell line. The shRNA-encoding plasmid was transfected into 293T cells together with the packaging plasmid using Trans-Lenticular TM Packaging System (Open Biosystems). On the next day, the growth medium was changed, and the supernatant containing lentivirus was collected and concentrated by centrifugation.
- pTRIPZ Open Biosystems
- V L ⁇ W 2/ 2 (V is the volume (mm 3), L is the maximum diameter (mm), W is the minimum diameter (mm)).
- mice were sacrificed according to standard protocols.
- p300 knockdown did not affect the growth of non-cancerous fibroblast cell line MRC5, immortalized renal epithelial cell line HEK293T, and immortalized retinal epithelial cell RPE-1 expressing CBP and p300 ( Figure 2).
- non-targeted shRNA or shp300 is expressed by a tetracycline-induced shRNA expression system using CBP mutant type LK2 cells.
- Cells were prepared and transplanted subcutaneously into nude mice. After the transplanted tumor became 200 mm 3 or more in size, in order to induce RNAi, doxycline was administered to the mouse, and the growth of the tumor was measured over time.
- LK2 shp300 xenografts was significantly suppressed in Dox-treated mice, but not significantly suppressed in LK2 shNT xenografts. This supports the synthetic lethal relationship between CBP and p300 in vivo.
- c-Myc was used as a marker for confirming the effect of the p300 inhibitor on cells in which CBP function was suppressed. It was considered possible.
- the present invention provides a therapeutic strategy for specifically targeting cancer cells in which the function of CBP is suppressed.
- CBP and p300 are in a synthetic lethal relationship, and it has been found that a treatment that inhibits p300 is a promising approach for the treatment of cancer in which the function of CBP is suppressed.
- this treatment strategy it was clarified that cancer patients can be selected using CBP function suppression as an index and then a p300 inhibitor can be administered, so that efficient treatment based on companion diagnosis is possible. . Therefore, the present invention can greatly contribute in the medical field, particularly in the field of cancer treatment.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
(a)CBP遺伝子に特異的に結合するオリゴヌクレオチドプライマー
(b)CBP遺伝子に特異的に結合するオリゴヌクレオチドプローブ
(c)CBPタンパク質に特異的に結合する抗体
(5)CBPの機能抑制が生じているがんの治療に用いる化合物のスクリーニング方法であって、p300を阻害するか否かを指標として化合物を選別する工程を含む方法。
本発明において、腫瘍、悪性腫瘍、がん、悪性新生物、がん腫、肉腫等を総称して、「腫瘍」または「がん」と表現する。本発明において、CBPとp300ががん細胞において合成致死の関係にあり、CBPの機能抑制が生じているがん細胞において、p300を阻害すると、当該がん細胞の増殖を抑制しうることが見出された。この知見に基づけば、CBPの機能抑制を指標として、p300を阻害する化合物によるがんの治療への応答性を評価することができる。従って、本発明は、がん患者由来の生物学的試料を用い、該生物学的試料中に含まれるCBPの機能抑制の有無を検出し、CBPの機能抑制が検出された患者を、p300を阻害する化合物によるがんの治療への応答性があると判定することを含む、p300を阻害する化合物によるがんの治療への応答性を予測する方法を提供する。
本発明において「変異を検出する」とは、原則として、ゲノムDNA上の変異を検出することを意味するが、当該ゲノムDNA上の変異が転写産物における塩基の変化や翻訳産物におけるアミノ酸の変化に反映される場合には、これら転写産物や翻訳産物における当該変化を検出すること(即ち、間接的な検出)をも含む意味である。
本発明における「CBPの発現低下」とは、通常、対照(例えば、健常者や同一患者の非がん組織における発現レベル)との比較において、それよりも発現レベルが低いことを意味する。
遺伝子の発現低下は、プロモーターの過剰メチル化が要因の一つであることが当該技術分野で公知である。従って、CBPの機能抑制の有無の検出においては、CBP遺伝子プロモーターのメチル化を指標として検出することも考えられる。プロモーターのメチル化の検出には、例えば、メチル化されたシトシンをウラシルに変換する活性を有するバイサルファイト処理後の塩基配列の変化を塩基配列決定により直接的に検出する方法や、バイサルファイト処理前の塩基配列は認識できる(切断できる)がバイサルファイト処理後の塩基配列は認識できない(切断できない)制限エンドヌクレアーゼを利用して間接的に検出する方法などの公知の方法を利用することができる。
また、本発明は、がん患者由来の生物学的試料を用い、該生物学的試料中に含まれるCBPの機能抑制の有無を検出し、CBPの機能抑制が検出された患者に対し、p300を阻害する化合物を投与することを含む、CBPの機能抑制が生じているがんの治療方法を提供する。
また、本発明は、上記の方法において、CBPの機能抑制の有無を検出するための試薬であって、下記の(a)~(c)のいずれかに記載の分子を有効成分とする試薬を提供する。
(a)CBP遺伝子に特異的に結合するオリゴヌクレオチドプライマー
(b)CBP遺伝子に特異的に結合するオリゴヌクレオチドプローブ
(c)CBPタンパク質に特異的に結合する抗体
上記ポリヌクレオチドプライマーは、CBPゲノムDNAやcDNAの塩基配列情報(例えば、配列番号:1や2)に基づき、上記した手法や増幅する領域に即したプライマーとなるように、また、CBP遺伝子以外の遺伝子の増幅産物が極力生じないように設計すればよい。このようなオリゴヌクレオチドプライマー設計は、当業者であれば、常法により行うことができる。オリゴヌクレオチドプライマーの長さは、通常15~50塩基長、好ましくは15~30塩基長であるが、手法および目的によってはこれより長くてもよい。
また、本発明は、CBPの機能抑制が生じているがんの治療に用いる化合物のスクリーニング方法であって、p300を阻害するか否かを指標として化合物を選別する工程を含む方法を提供する。
1.材料および方法
(1)細胞株
A549、H1299、H157、SQ5、H1703、LK2、およびH520(NSCLC)、RL、Loucy、RC-K8、U2932、Ramos、Farage、SUP-T1、WSU-NHL、VAL、SUDHL5、Jurkat、TE8、TE10を10%のウシ胎児血清(FBS)を添加したRPMI-1640またはDMEM中で培養した。MRC-5細胞(正常線維芽細胞)、HEK293T細胞(不死化腎上皮細胞)、RPE-1細胞(不死化網膜上皮細胞)を10% FBSを添加したDMEM中で培養した。
各種タンパク質のノックダウンには、ON-TARGET plus SMARTpool siRNA(Dharmacon社)を用いた。トランスフェクションにはLipofectamine RNAiMAX(Invitrogen社)を用いた。非標的化siRNA(L-001810-10)を陰性対照として用いた。
以下のタンパク質に対して特異的な抗体を用いて、文献(Ogiwara H, et al. Oncogene 2011;5;30:2135-46)に記載されたように、イムノブロッティングを実施した。CBP(Santacruz社;sc-369X)、p300(Santacruz社;sc-48343X)、H3(Active motif社;39163)、H3K18ac(Millipore社;07-354)、β-actin(Cell signaling Technologies社;4970)、cleaved PARP(Cell signaling Technologies社;5625)、p21/CDKN1A(Cell Signaling Technologies社;2947)、LC3B(Cell Signaling Technologies社;3868)。
がん細胞の生存に及ぼすsiRNAノックダウンの影響を、クロノゲニック生存アッセイ(clonogenic survival assays)を用いて評価した。Lipofectamine RNAiMAX(Invitrogen社)を用いてがん細胞株にsiRNAs(50nM)をトランスフェクションした。2日後に細胞をトリプシン処理し、カウントし、6ウェルの培養皿に特定の数で再度播種し、さらに12日間(各種HATのノックダウンにおいては14日間)培養してコロニーを形成させた。次いで、細胞を50%(v/v)メタノール/0.01%(w/v)クリスタルバイオレットを含む溶液中で5分間固定しコロニー数をカウントした。
生存率の評価は、CellTiter-Glo Luminescent Cell Viability Assay kit(Promega社)を使用して、細胞内ATPレベルを測定することによって決定した。Lipofectamine RNAiMAX(Invitrogen社)を用いてがん細胞株にsiRNAs(50nM)をトランスフェクションした。2日後に細胞をトリプシン処理し、カウントし、96ウェルプレートに特定の数で再度播種した。細胞の生存率を測定するために、CellTiter-Glo Luminescent Cell Viability Assay kit(Promega社)を細胞に添加し、Envision(PerkinElmer社)で蛍光を測定した。
細胞をトリプシン処理し、遠心分離し、PBSで洗浄して、氷冷した70%エタノールで固定化した。次いで、細胞を再度遠心分離し、200μg/mlのRNアーゼAおよび5μg/mlのヨウ化プロピジウムを含有するPBSでインキュベートして、Guava flow cytometry(Millipore社)で細胞周期分布を分析した。
フローサイトメトリーによりアポトーシス細胞を検出するために、Annexin V-FITC/PI apoptosis detection kit(Roche社)をメーカーの使用説明書に従って用いた。すなわち、細胞ペレットを1×binding buffer中に懸濁し、暗中で20分間Annexin V-FITCおよびPIとインキュベートした。次いで、細胞の蛍光をフローサイトメトリーで分析した。
tet誘導性細胞株を作製するために、細胞株にshRNA発現レンチウイルスベクター由来のpTRIPZ(Open Biosystems社)をトランスダクションした。shRNAをコードするプラスミドをTrans-LentiviralTM Packaging System(Open Biosystems社)を用いてパッケージングプラスミドとともに293T細胞にトランスフェクションした。翌日に増殖培地を交換し、レンチウイルスを含む上清を回収し、遠心分離により濃縮した。
tet誘導性の細胞株であるLK2-shp300細胞およびLK2-shNT細胞(50% Matrigel中で2×106細胞/マウス;BD Biosciences社)をカウントし、氷上で1:1の培地/Matrigel(BD Bioscience社)で再懸濁し、国立がん研究センターの実験動物に関する倫理委員会によって承認されたプロトコールで、7週齢の雌BALB/c-nu/nuマウス(CLEA Japan社)の脇腹の皮下に注入した。3週間後、腫瘍が200mm3以上の大きさに到達したとき、マウスを無作為に2群に分け、doxycycline(200 ppm)を含有する餌または対照餌のいずれかを与えた。腫瘍の成長を、ノギスを使用して1週間に2回測定した。移植した腫瘍の体積は、ノギスを使用して3~4日毎に、次の式で計算した。式:V=L×W2/2(Vは体積(mm3)、Lは最大直径(mm)、Wは最小直径(mm))。実験の最後に、標準的なプロトコールに従って、マウスを屠殺した。
全ての実験を三重実験で行った。データは、平均±SDで示した。薬剤処理した細胞と非処理の細胞との差は、スチューデントのt検定で評価し、統計的有意差をアスタリスクで示した(「*」はP<0.05を、「**」はP<0.01を、「***」はP<0.001を、「****」はP<0.0001をそれぞれ示す)。
(1)CBP変異型がん細胞のp300依存性増殖
我々は、細胞増殖アッセイおよびクロノゲニック生存アッセイを用いて、siRNAを介したp300消失が、CBP野生型がん細胞株およびCBP変異型がん細胞株の増殖に与える影響を比較した(図1A~C)。その結果、検証した全てのCBP変異型細胞において細胞増殖とコロニー形成の抑制が観察されたが、これはCBP野生型細胞株では観察されなかった。
我々は、次に、CBP変異型の肺がん細胞およびリンパ腫細胞に対するp300阻害剤C646の影響を検証した。その結果、CBP変異型がん細胞がCBP野生型がん細胞よりもC646に対して高い感受性を有することを観察した(図3A,C)。C646のCBP変異型がん細胞に対するIC50値は、CBP野生型がん細胞に対するIC50値と比較して低い傾向にあった(スチューデントのt検定でp<0.01)(図3B,D)。これらの結果は、p300のヒストンアセチルトランスフェラーゼ活性の阻害が、CBP変異型がん細胞の致死効果を特異的に引き起こすことを示唆した。
我々は、次に、p300の消失や阻害によるCBP変異型がん細胞の増殖阻害のメカニズムについて検証した。CBP変異型H1703細胞におけるsiRNAを介したp300消失は、分解されたPARP(アポトーシスのバイオマーカー)の量を増加させたが、p21/CDKN1A(細胞老化のバイオマーカー)やLC3B(オートファジーのバイオマーカー)の量は増加させなかった(図4)。これと一致して、アネキシンVで染色した細胞のフローサイトメトリー分析により、siRNAによるp300の消失が、CBP変異型のH1703細胞およびLK2細胞において、アネキシンV陽性アポトーシス細胞の割合を増加させるが、CBP野生型のH157細胞やSQ5細胞では増加させないことが確認された(図5A)。
in vivo前臨床バリデーションモデルとして、CBP変異型LK2細胞を用いて、テトラサイクリン誘導shRNA発現システムにより、非標的化shRNAあるいはshp300を発現する細胞を作製し、ヌードマウスの皮下に移植した。移植腫瘍が200mm3以上の大きさになった後、RNAiを誘導するために、マウスにdoxycyclineを投与し、経時的に腫瘍の増殖を測定した。図6に示すように、LK2 shp300異種移殖片の増殖は、Dox処理したマウスで有意に抑制されたが、LK2 shNT異種移殖片では有意に抑制されなかった。このことは、in vivoにおけるCBPとp300との合成致死の関係を裏付けるものである。
A549細胞(CBPは野生型)およびH520細胞(CBPは変異型)における各種HATのノックダウンによる細胞生存率へ影響を観察した。A549細胞においては各種HATのノックダウンにより細胞生存率への影響は見られなかったが、H520細胞においてはp300のノックダウンにより有意な細胞生存率低下が確認された(図8)。
各種細胞におけるp300ノックダウン時のc-Mycタンパク質発現の変化を観察した。
Claims (6)
- がん患者由来の生物学的試料を用い、該生物学的試料中に含まれるCBPの機能抑制の有無を検出し、CBPの機能抑制が検出された患者を、p300を阻害する化合物によるがんの治療への応答性があると判定することを含む、p300を阻害する化合物によるがんの治療への応答性を予測する方法。
- がん患者由来の生物学的試料を用い、該生物学的試料中におけるCBPの機能抑制の有無を検出し、CBPの機能抑制が検出された患者を、p300を阻害する化合物によるがんの治療の対象として選別することを含む、p300を阻害する化合物によるがんの治療の対象を選別する方法。
- がん患者由来の生物学的試料を用い、該生物学的試料中に含まれるCBPの機能抑制の有無を検出し、CBPの機能抑制が検出された患者に対し、p300を阻害する化合物を投与することを含む、CBPの機能抑制が生じているがんの治療方法。
- 請求項1から3のいずれかに記載の方法において、CBPの機能抑制の有無を検出するための試薬であって、下記の(a)~(c)のいずれかに記載の分子を有効成分とする試薬。
(a)CBP遺伝子に特異的に結合するオリゴヌクレオチドプライマー
(b)CBP遺伝子に特異的に結合するオリゴヌクレオチドプローブ
(c)CBPタンパク質に特異的に結合する抗体 - CBPの機能抑制が生じているがんの治療に用いる化合物のスクリーニング方法であって、p300を阻害するか否かを指標として化合物を選別する工程を含む方法。
- p300を阻害する化合物を含む、CBPの機能抑制が検出されたがんの治療剤。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ES15752232T ES2773456T3 (es) | 2014-02-24 | 2015-02-23 | Método para predecir la capacidad de respuesta al tratamiento del cáncer usando un compuesto que inhibe p300 |
JP2016504207A JP6588006B2 (ja) | 2014-02-24 | 2015-02-23 | p300を阻害する化合物によるがんの治療への応答性を予測する方法 |
EP15752232.7A EP3121274B1 (en) | 2014-02-24 | 2015-02-23 | METHOD FOR PREDICTING RESPONSIVENESS TO CANCER TREATMENT USING p300-INHIBITING COMPOUND |
US15/121,001 US10768179B2 (en) | 2014-02-24 | 2015-02-23 | Method for predicting responsiveness to cancer treatment using p300-inhibiting compound |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014-032928 | 2014-02-24 | ||
JP2014032928 | 2014-02-24 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2015125956A1 true WO2015125956A1 (ja) | 2015-08-27 |
Family
ID=53878452
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/054991 WO2015125956A1 (ja) | 2014-02-24 | 2015-02-23 | p300を阻害する化合物によるがんの治療への応答性を予測する方法 |
Country Status (5)
Country | Link |
---|---|
US (1) | US10768179B2 (ja) |
EP (1) | EP3121274B1 (ja) |
JP (1) | JP6588006B2 (ja) |
ES (1) | ES2773456T3 (ja) |
WO (1) | WO2015125956A1 (ja) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110170052B (zh) * | 2019-06-21 | 2020-07-10 | 复旦大学 | Cbp-p300抑制剂在肠道损伤疾病中的应用 |
CN116904469B (zh) * | 2023-09-12 | 2024-01-23 | 首都儿科研究所 | 一种p300蛋白表达的抑制剂和制备方法及其用途 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008157680A2 (en) * | 2007-06-21 | 2008-12-24 | The Wistar Institute | Methods and compositions for modulating p300/cbp activity |
WO2011085039A2 (en) * | 2010-01-05 | 2011-07-14 | The Johns Hopkins University | Use of histone acetyltransferase inhibitors as novel anti-cancer therapies |
WO2015005473A1 (ja) * | 2013-07-12 | 2015-01-15 | 独立行政法人国立がん研究センター | がんの治療への応答性を予測する方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013148114A1 (en) | 2012-03-30 | 2013-10-03 | University Of Florida Research Foundation, Inc. | P300/cbp inhibitors and methods of use |
-
2015
- 2015-02-23 EP EP15752232.7A patent/EP3121274B1/en active Active
- 2015-02-23 ES ES15752232T patent/ES2773456T3/es active Active
- 2015-02-23 US US15/121,001 patent/US10768179B2/en active Active
- 2015-02-23 JP JP2016504207A patent/JP6588006B2/ja active Active
- 2015-02-23 WO PCT/JP2015/054991 patent/WO2015125956A1/ja active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008157680A2 (en) * | 2007-06-21 | 2008-12-24 | The Wistar Institute | Methods and compositions for modulating p300/cbp activity |
WO2011085039A2 (en) * | 2010-01-05 | 2011-07-14 | The Johns Hopkins University | Use of histone acetyltransferase inhibitors as novel anti-cancer therapies |
WO2015005473A1 (ja) * | 2013-07-12 | 2015-01-15 | 独立行政法人国立がん研究センター | がんの治療への応答性を予測する方法 |
Non-Patent Citations (7)
Title |
---|
CHAN D.A. ET AL.: "Harnessing synthetic lethal interactions in anticancer drug discovery.", NAT. REV. DRUG. DISCOV., vol. 10, no. 5, 2011, pages 351 - 364, XP055204267, ISSN: 1474-1776 * |
KISHIMOTO M. ET AL.: "Mutations and deletions of the CBP gene in human lung cancer.", CLIN. CANCER RES., vol. 11, 2005, pages 512 - 519, XP055221483 * |
OGIWARA H. ET AL.: "CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.", PLOS ONE, vol. 7, no. 12, 2012, pages e52810, XP055221486 * |
OGIWARA H. ET AL.: "Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors.", ONCOGENE, vol. 30, no. 18, 2011, pages 2135 - 2146, XP055221484, ISSN: 0950-9232 * |
OIKE T. ET AL.: "Chromatin-regulating proteins as targets for cancer therapy.", J. RADIAT. RES., vol. 55, no. 4, 11 February 2014 (2014-02-11), pages 613 - 628, XP055221487, ISSN: 0449-3060 * |
See also references of EP3121274A1 * |
TAKASHI KONO: "Gan no Kobetsuka Chiryo to Soyaku", JAPANESE JOURNAL OF CLINICAL MEDICINE, vol. 72, no. suppl.2, 20 February 2014 (2014-02-20), pages 34 - 39 * |
Also Published As
Publication number | Publication date |
---|---|
ES2773456T3 (es) | 2020-07-13 |
EP3121274A4 (en) | 2017-10-18 |
EP3121274A1 (en) | 2017-01-25 |
US10768179B2 (en) | 2020-09-08 |
JP6588006B2 (ja) | 2019-10-09 |
EP3121274B1 (en) | 2019-12-18 |
US20170067899A1 (en) | 2017-03-09 |
JPWO2015125956A1 (ja) | 2017-03-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10221459B2 (en) | Compositions and methods of treating cancer harboring PIKC3A mutations | |
WO2015005473A1 (ja) | がんの治療への応答性を予測する方法 | |
US20140243378A1 (en) | Compositions and Methods for the Treatment of Muscular Disease, and Related Screening Methods | |
US20090209618A1 (en) | Pyruvate dehydrogenase kinases as therapeutic targets for cancer and ischemic diseases | |
US11135218B2 (en) | Synthetic lethality and the treatment of cancer | |
Park et al. | HDAC6 sustains growth stimulation by prolonging the activation of EGF receptor through the inhibition of rabaptin-5-mediated early endosome fusion in gastric cancer | |
Han et al. | Long noncoding RNA TUG1 regulates degradation of chondrocyte extracellular matrix via miR-320c/MMP-13 axis in osteoarthritis | |
US20190203304A1 (en) | Method for predicting responsiveness to phosphatidylserine synthase 1 inhibitor | |
JP6588006B2 (ja) | p300を阻害する化合物によるがんの治療への応答性を予測する方法 | |
US20060216731A1 (en) | Methods and reagents for diagnosing and treating gliomas | |
Zou et al. | Downregulation of circ_0037655 impedes glioma formation and metastasis via the regulation of miR-1229-3p/ITGB8 axis | |
KR20230088634A (ko) | 간암 특이적 바이오 마커 및 이의 용도 | |
Liu et al. | Circ_WWC3 overexpression decelerates the progression of osteosarcoma by regulating miR-421/PDE7B axis | |
US20230272478A1 (en) | Biomarker specific for liver cancer, and use thereof | |
JP5592793B2 (ja) | Dnaメチル化阻害剤の薬剤効果検出方法 | |
JP6839707B2 (ja) | Gpr160を過剰発現する癌の予防、診断および治療 | |
WO2021100869A1 (ja) | ヘリカーゼ阻害剤に対するがん細胞の感受性を予測する方法 | |
KR20190005727A (ko) | 난소과립막세포암 또는 자궁내막암의 진단 및 치료를 위한 마이크로rna-1236의 용도 | |
WO2009084668A1 (ja) | 癌細胞増殖阻害方法、増殖阻害剤及びスクリーニング方法 | |
Zhang et al. | PIMT is a novel and potent suppressor of endothelial activation | |
WO2024003350A1 (en) | Combination therapy for melanoma | |
Lu et al. | Long Non-Coding RNA SNHG19 Promotes Breast Cancer Progression by Regulating miR-299-5p | |
KR20230045386A (ko) | miR-4487를 유효성분으로 함유하는 항암제 민감성 증진용 조성물 | |
WO2021094426A1 (en) | Method for determining parp inhibitor responsiveness, and improved parp inhibitor therapy | |
JP2011111435A (ja) | 神経芽腫又は膠芽腫の治療薬 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15752232 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2016504207 Country of ref document: JP Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15121001 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2015752232 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2015752232 Country of ref document: EP |