Patent Application
20170067899
The present invention relates to a method for predicting responsiveness to the treatment of cancer with a compound inhibiting p300 and a method for selecting a candidate for the treatment of cancer by using functional suppression of CBP as an index. The present invention also relates to a method for treating cancer having functional suppression of CBP with a compound inhibiting p300. The present invention further relates to a reagent for detecting the presence or absence of functional suppression of CBP, which is used in these methods. The present invention further relates to a method for screening for a compound for use in the treatment of cancer having functional suppression of CBP by using inhibition of p300 as an index.
Tyrosine kinase inhibitors are effective against solid tumors with activating mutations in tyrosine kinase gene, such as EGFR mutations or ALK fusion found in lung adenocarcinoma (NPL 1). We and other researchers have recently identified RET oncogene fusions in lung adenocarcinoma (NPL 2). This supports the importance of tyrosine kinase gene as a therapeutic target.
Meanwhile, inactivating somatic mutations in genes encoding chromatin-regulating protein subunits (e.g., histone acetyltransferases CBP/CREBBP and p300/EP300, histone methyltransferases MLL2 and SETD2, histone demethylases JARID1C and UTX, and chromatin remodeling factors BRG1, ARID1A, ARID2, PBRM1, and SNF5) have been largely attractive since they were first identified by the genome-wide sequencing analysis of cancer cells. These mutations are considered to inhibit functions in transcription or DNA double-strand break repair and thought to be crucial to the development and/or progression of cancer.
Unfortunately, therapeutic strategies for specifically targeting cancer cells having these mutations have not yet been developed.
[NPL 1] Pao W, Girard N. Lancet Oncol. 2011; February; 12 (2): 175-80 [NPL 2] Shaw A T, et al., Nat Rev Cancer. 2013; November; 13 (11): 772-87
The present invention has been made in light of these circumferences, and an object of the present invention is to develop a therapeutic strategy for specifically targeting cancer cells having functional suppression of CBP.
Synthetic lethality therapy is a very promising method for treating cancer. For example, BRCA1 and BRCA2 genes are in the relationship of synthetic lethality with PARP1 gene. The growth of BRCA1-deficient or BRCA2-deficient cancer cells depends on the functions of the PARP1 protein. Now, these findings have been translated to the clinic through the development of PARP inhibitors to treat BRCA1/BRCA2-deficient tumors (Chan D A, et al., Nat Rev Drug Discov. 2011; 10: 351-64). In addition, the synthetic lethality therapy has been proposed for the treatment of cancer deficient in a gene involved in DNA mismatch repair or cell metabolism (Muller F L, et al., Nature. 2012; 488: 337-42, Chan D A, et al., Sci Transl Med. 2011; 3: 94ra70, and Martin S A, et al., Cancer Cell. 2010; 17: 235-48). The present inventors have conducted diligent studies in order to apply the synthetic lethality therapy to a therapeutic strategy for specifically killing cancer cells harboring CBP mutations.
As a result, the present inventors have found that suppression of p300 protein expression or functional inhibition of this protein in cancer cells harboring CBP mutations remarkably suppresses the growth of the cancer cells, whereas such suppression of growth does not occur in CBP-proficient cells. Also, the percentage of cells positive to Annexin V/PI staining was increased in cancer cells whose growth was suppressed, demonstrating that apoptosis was induced. Furthermore, an experiment using mice in which cancer cells harboring CBP mutations were transplanted has also demonstrated the in vivo inhibitory effect of suppression of p300 expression on the growth of cancer cells harboring CBP mutations.
From these results, the present inventors have found that CBP and p300 are in the relationship of synthetic lethality, and treatment inhibiting p300 (particularly, its histone acetyltransferase activity) is a promising approach for the treatment of cancer having functional suppression of CBP. The present inventors have also revealed that this therapeutic strategy achieves efficient treatment based on companion diagnostics because a p300 inhibitor can be administered to a cancer patient selected with functional suppression of CBP as an index.
The present inventors have further found that screening for a drug useful in the treatment of CBP-mutated cancer can be performed by using whether or not to inhibit p300 as an index. On the basis of these findings, the present invention has been completed.
Thus, the present invention relates to synthetic lethality therapy for specifically targeting cancer cells having functional suppression of CBP such as CBP mutations, and companion diagnostics for the synthetic lethality therapy, and more specifically provides the following aspects:
(1) A method for predicting responsiveness to the treatment of cancer with a compound inhibiting p300, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP contained in the biological sample, and determining the patient with the detected functional suppression of CBP as responsive to the treatment of cancer with the compound inhibiting p300.
(2) A method for selecting a candidate for the treatment of cancer with a compound inhibiting p300, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP in the biological sample, and selecting the patient with the detected functional suppression of CBP as the candidate for the treatment of cancer with the compound inhibiting p300.
(3) A method for treating cancer having functional suppression of CBP, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP contained in the biological sample, and administering a compound inhibiting p300 to the patient with the detected functional suppression of CBP.
(4) A reagent for detecting the presence or absence of functional suppression of CBP in a method according to any of (1) to (3), the reagent comprising any of the following molecules (a) to (c) as an active ingredient:
(a) an oligonucleotide primer specifically binding to the CBP gene,
(b) an oligonucleotide probe specifically binding to the CBP gene, and
(c) an antibody specifically binding to the CBP protein.
(5) A method for screening for a compound for use in the treatment of cancer having functional suppression of CBP, the method comprising the step of selecting the compound by using whether or not to inhibit p300 as an index.
(6) A therapeutic agent for cancer in which functional suppression of CBP has been detected, comprising a compound inhibiting p300.
According to the present invention, responsiveness to the treatment of cancer with a p300 inhibitor can be efficiently predicted with functional suppression of CBP as an index. According to the present invention, the presence or absence of functional suppression of CBP (e.g., inactivating mutations or reduction in expression) in a biological sample derived from a cancer patient is detected, and the patient having the functional suppression of CBP is selected. Then, this selected patient can be subjected to the treatment of cancer with a p300 inhibitor. Therefore, treatment results of cancer patients can be largely improved. Use of a probe or a primer for the CBP gene and an antibody against CBP allows companion diagnostics to be efficiently performed by such detection of the presence or absence of functional suppression of CBP.
<Method for Predicting Responsiveness to Treatment of Cancer and Method for Selecting Candidate for Treatment of Cancer>
In the present invention, tumor, malignant tumor, cancer, malignant neoplasm, carcinoma, sarcoma, and the like are collectively referred to as “tumor” or “cancer”. In the present invention, it has been found that CBP and p300 are in the relationship of synthetic lethality in cancer cells, and inhibition of p300 in cancer cells having functional suppression of CBP can suppress the growth of the cancer cells. On the basis of this finding, responsiveness to the treatment of cancer with a compound inhibiting p300 can be evaluated with functional suppression of CBP as an index. Thus, the present invention provides a method for predicting responsiveness to the treatment of cancer with a compound inhibiting p300, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP contained in the biological sample, and determining the patient with the detected functional suppression of CBP as responsive to the treatment of cancer with the compound inhibiting p300.
Such a patient with the detected functional suppression of CBP is suitable for the treatment of cancer with the compound inhibiting p300. Efficient treatment can be performed by selecting a patient responsive to and a patient nonresponsive to the treatment of cancer with the compound inhibiting p300 with functional suppression of CBP as an index. Thus, the present invention provides even a method for selecting a candidate for the treatment of cancer with a compound inhibiting p300, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP in the biological sample, and selecting the patient with the detected functional suppression of CBP as the candidate for the treatment of cancer with the compound inhibiting p300.
In the present invention, the “cancer patient” may be not only a human affected by cancer but a human suspected of having cancer. In the method of the present invention, the cancer patient in which the presence or absence of functional suppression of CBP is to be detected is not particularly limited, and every cancer patient can be used as this subject. The cancer found to have functional suppression of CBP may be, for example, lung cancer, bladder cancer, lymphoma, or adenoid cystic cancer. A 10% subset of lung cancer, a 13% subset of bladder cancer, a 20 to 40% subset of lymphoma, and a 7% subset of adenoid cystic cancer are known to have CBP mutations. Therefore, according to the method of the present invention, a cancer patient having such a frequency can be selected as the “candidate for the treatment”.
The “biological sample” used in the present invention is not particularly limited as long as the presence or absence of functional suppression of CBP can be detected in the biological sample. A cancer biopsy specimen is preferred. Protein extracts or nucleic acid extracts (mRNA extracts, cDNA or cRNA preparations prepared from the mRNA extracts, etc.) obtained from these samples may be used.
In the present invention, both “CBP” and “p300” are histone acetyltransferases that are involved in chromatin regulation. These enzymes are in a paralogous relationship. The typical nucleotide sequence of human-derived natural CBP genomic DNA is shown in SEQ ID NO: 1. The typical nucleotide sequence of human-derived natural CBP cDNA is shown in SEQ ID NO: 2. The typical amino acid sequence of human-derived natural CBP protein is shown in SEQ ID NO: 3. The typical nucleotide sequence of human-derived natural p300 genomic DNA is shown in SEQ ID NO: 4. The typical nucleotide sequence of human-derived natural p300 cDNA is shown in SEQ ID NO: 5. The typical amino acid sequence of human-derived natural p300 protein is shown in SEQ ID NO: 6. It should be understood that even CBP or p300 having no mutation may vary in sequence among individuals due to polymorphism, etc.
In the present invention, the “functional suppression of CBP” includes both of inactivation of CBP or reduction in CBP activity and reduction in CBP expression. The inactivation of CBP is typically attributed to an inactivating mutation in CBP. The inactivating mutation may occur due to, for example, a missense mutation in the histone acetyltransferase (HAT) domain (1342- to 1648-positions in the amino acid sequence represented by SEQ ID NO: 3) of CBP, a nonsense mutation over the whole region, or complete or partial deletion of the gene. The inactivating mutation is not limited thereto as long as the mutation causes inactivation of CBP. Examples of the CBP mutations are shown in Table 1 and
The reduction in CBP expression includes both of reduction in expression at the transcriptional level and reduction in expression at the translational level.
In the present invention, examples of the approach for “detecting functional suppression of CBP” include, but are not particularly limited to, methods described below.
—Detection of CBP Mutation—
In the present invention, the phrase “detecting a mutation” means to detect a mutation in genomic DNA as a rule and includes even to detect change in base in a transcription product or change in amino acid in a translation product (i.e., indirect detection) when the mutation in genomic DNA is reflected to such change in the transcription product or the translation product.
A preferred embodiment of the method of the present invention is a method for detecting a mutation by directly determining the nucleotide sequence of a CBP gene region in a cancer patient. In the present invention, the “CBP gene region” means a given region in the genomic DNA containing the CBP gene. This region also includes the expression control regions (e.g., a promoter region and an enhancer region) of the CBP gene, the 3′-untranslated region of the CBP gene, and the like. Mutations in these regions may influence, for example, the transcriptional activity of the CBP gene.
In this method, first, a DNA sample is prepared from a biological sample derived from a cancer patient. Examples of the DNA sample include a genomic DNA sample and a cDNA sample prepared from RNA by reverse transcription.
The method for extracting genomic DNA or RNA from the biological sample is not particularly limited and can be appropriately selected for use from approaches known in the art. Examples of the method for extracting the genomic DNA include SDS phenol method (method which involves denaturing proteins of a tissue preserved in a urea-containing solution or ethanol using a proteolytic enzyme (proteinase K), a surfactant (SDS), and phenol, and precipitating and extracting DNA from the tissue with ethanol), and DNA extraction method using Clean Columns® (manufactured by Hermes-NexTec GmbH), AquaPure® (manufactured by Bio-Rad Laboratories, Inc.), ZR Plant/Seed DNA Kit (manufactured by Zymo Research Corp.), AquaGenomic Solution® (manufactured by MoBiTec GmbH), prepGEM® (manufactured by ZyGEM NZ, Ltd.), or BuccalQuick® (manufactured by TrimGen Corp.). Examples of the method for extracting the RNA include extraction method using phenol and a chaotropic salt (more specifically, extraction method using a commercially available kit such as TRIzol (manufactured by Invitrogen Corp.) or IISOGEN® (manufactured by Wako Pure Chemical Industries, Ltd.)), and methods using other commercially available kits (RNAPrep total RNA extraction kit (manufactured by Beckman Coulter Inc.), RNeasy Mini (manufactured by Qiagen N.V.), RNA Extraction Kit (manufactured by Pharmacia Biotech Inc.), etc.). Examples of the reverse transcriptase for use in preparing cDNA from the extracted RNA include, but are not particularly limited to, reverse transcriptase derived from retrovirus such as RAV (Rous associated virus) or AMV (avian myeloblastosis virus), and reverse transcriptase derived from mouse retrovirus such as MMLV (Moloney murine leukemia virus).
In this embodiment, subsequently, DNA containing a mutation site in the CBP gene region is isolated, and the nucleotide sequence of the isolated DNA is determined. The isolation of the DNA can be carried out by, for example, PCR with the genomic DNA or the RNA as a template using a pair of oligonucleotide primers designed to flank the mutation site in the CBP gene region. The determination of the nucleotide sequence of the isolated DNA can be carried out by a method generally known to those skilled in the art, such as Maxam-Gilbert method or Sanger method.
The determined nucleotide sequence of the DNA or the cDNA can be compared with a control (e.g., the nucleotide sequence of DNA or cDNA derived from a noncancerous tissue of the same patient) to determine the presence or absence of a mutation in the CBP gene region in the cancer cells of the cancer patient.
The method for detecting a mutation in the CBP gene region can be carried out by various methods capable of detecting mutations, in addition to the method for directly determining the nucleotide sequence of DNA or cDNA.
The detection of a mutation according to the present invention can also be carried out by, for example, the following method: first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, a reporter fluorescent dye- and quencher fluorescent dye-labeled oligonucleotide probe having a nucleotide sequence complementary to a nucleotide sequence containing the mutation in the CBP gene region is prepared. Then, the oligonucleotide probe is hybridized to the DNA sample, and the nucleotide sequence containing the mutation in the CBP gene region is further amplified with the DNA sample hybridized with the oligonucleotide probe as a template. The fluorescence emitted by the reporter fluorescent dye as a result of degradation of the oligonucleotide probe by the amplification is detected. Subsequently, the detected fluorescence is compared with the control. Examples of such a method include double dye probe method, so-called TaqMan® probe method.
In a further alternative method, a DNA or cDNA sample is prepared from the biological sample. Subsequently, a nucleotide sequence containing the mutation in the CBP gene region is amplified with the DNA sample as a template in a reaction system containing an intercalator that emits fluorescence when inserted to between DNA double strands. The temperature of the reaction system is changed, and variation in the intensity of fluorescence emitted by the intercalator is detected. The detected variation in the fluorescence intensity caused by the change in the temperature is compared with the control. Examples of such a method include HRM (high resolution melting) method.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. Further, the amplified DNA is cleaved with restriction enzymes. Subsequently, the DNA fragment is separated according to its size.
Subsequently, the detected size of the DNA fragment is compared with the control. Examples of such a method include a method based on restriction fragment length polymorphism (RFLP) and PCR-RFLP.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. Further, the amplified DNA is dissociated into single-stranded DNA. Subsequently, the single-stranded DNA thus obtained by dissociation is separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with the control. Examples of such a method include PCR-SSCP (single-strand conformation polymorphism).
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. Further, the amplified DNA is separated on a gel in which the concentration of a DNA denaturant is gradually elevated. Subsequently, the mobility of the separated DNA on the gel is compared with the control. Examples of such a method include denaturant gradient gel electrophoresis (DGGE).
A further alternative method is a method using DNA containing the mutation site in the CBP gene region prepared from the biological sample, and a substrate with an immobilized oligonucleotide probe hybridizing to the DNA. Examples of such a method include DNA array method.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Also, an “oligonucleotide primer having a nucleotide sequence complementary to a base immediately 3′ to the base of the mutation site in the CBP gene region and a 3′-nucleotide sequence thereof” is prepared. Subsequently, ddNTP primer extension reaction is performed with the DNA as a template using the primer. Subsequently, the primer extension reaction product is subjected to mass spectrometry in a mass spectrometer. Subsequently, the genotype is determined from the results of mass spectrometry. Subsequently, the determined genotype is compared with the control. Examples of such a method include MALDI-TOF/MS.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, an oligonucleotide probe consisting of 5′-“a nucleotide sequence complementary to the base of the mutation site in the CBP gene region and a 5′-nucleotide sequence thereof“−” a nucleotide sequence that does not hybridize to a base immediately 3′ to the mutation site in the CBP gene region and a 3′-nucleotide sequence thereof”-3′ (flap) is prepared. Also, an “oligonucleotide probe having a nucleotide sequence complementary to the base of the mutation site in the CBP gene region and a 3′-nucleotide sequence thereof” is prepared. Subsequently, these two types of oligonucleotide probes are hybridized to the prepared DNA. Subsequently, the hybridized DNA is cleaved with a single-stranded DNA-cleaving enzyme to liberate the flap. Examples of the single-stranded DNA-cleaving enzyme include, but are not particularly limited to, cleavase. In this method, a fluorescence reporter- and fluorescence quencher-labeled oligonucleotide probe having a sequence complementary to the flap is then hybridized to the flap. Subsequently, the intensity of the generated fluorescence is measured. Subsequently, the measured fluorescence intensity is compared with the control. Examples of such a method include Invader method.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. The amplified DNA is dissociated into single-stranded DNA. Only one of the single DNA strands thus obtained by dissociation is separated. Subsequently, extension reaction is performed by one base at a time from near the base of the mutation site in the CBP gene region. Pyrophosphate generated during this reaction is enzymatically allowed to emit light, and the intensity of the luminescence is measured. Then, the measured fluorescence intensity is compared with the control. Examples of such a method include pyrosequencing method.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. Subsequently, an “oligonucleotide primer having a nucleotide sequence complementary to a base immediately 3′ to the base of the mutation site in the CBP gene region and a 3′-nucleotide sequence thereof” is prepared. Subsequently, single-nucleotide extension reaction is performed with the amplified DNA as a template using the prepared primer in the presence of a fluorescently labeled nucleotide. The degree of polarization of fluorescence is measured. Subsequently, the measured degree of polarization of fluorescence is compared with the control. Examples of such a method include AcycloPrime method.
In a further alternative method, first, a DNA or cDNA sample is prepared from the biological sample. Subsequently, DNA containing the mutation site in the CBP gene region is amplified. Subsequently, an “oligonucleotide primer having a nucleotide sequence complementary to a base immediately 3′ to the base of the mutation site in the CBP gene region and a 3′-nucleotide sequence thereof” is prepared. Subsequently, single-nucleotide extension reaction is performed with the amplified DNA as a template using the prepared primer in the presence of a fluorescently labeled nucleotide. Subsequently, the base species used in the single-nucleotide extension reaction is determined. Subsequently, the determined base species is compared with the control. Examples of such a method include SNuPE method.
Provided that the mutation involves change (e.g., substitution, deletion, or insertion) in amino acid in the CBP protein, the sample prepared from the biological sample may be the protein. In such a case, for example, a method using a molecule (e.g., an antibody) specifically binding to a site having change in amino acid caused by the mutation can be utilized for detecting the mutation. The method for detecting the protein using an antibody will be described later.
—Detection of Reduction in CBP Expression—
In the present invention, the “reduction in CBP expression” usually means a lower expression level as compared with a control (e.g., an expression level in the noncancerous tissue of a healthy person or the same patient).
In the method for detecting the CBP expression level at the transcriptional level, first, RNA or cDNA is prepared by the method described above from a biological sample derived from a cancer patient. Subsequently, an oligonucleotide primer and an oligonucleotide probe are used in amplification reaction and hybridization reaction, respectively, to detect the amplification product or the hybridization product. For example, RT-PCR, Northern blotting, dot blotting, DNA array method, in situ hybridization, RNase protection assay, or mRNA-seq can be used as such a method. Those skilled in the art can design an oligonucleotide primer or an oligonucleotide probe suitable for each method by a routine method on the basis of the nucleotide sequence (e.g., SEQ ID NO: 2) of CBP cDNA.
In the method for detecting the CBP expression level at the translational level, first, a protein sample is prepared from a biological sample derived from a cancer patient. Subsequently, an antibody specific for the CBP protein is used in antigen-antibody reaction to detect the binding of the antibody to the CBP protein. When the antibody specific for CBP is labeled, the CBP protein can be detected directly. When this antibody is not labeled, a labeled molecule (e.g., secondary antibody or protein A) that recognizes this antibody is further allowed to act on the antigen-antibody complex, and the CBP protein can be detected indirectly through the use of the label of the molecule. For example, immunohistochemical (immunostaining) method, Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, or analysis method using an antibody array can be used as such a method. The immunohistochemistry also has the advantage that additional information such as the morphology or distributed state of cancer cells in a tissue can be obtained at the same time.
The antibody used is not particularly limited by its type, origin, etc., and is preferably a monoclonal antibody. An oligoclonal antibody (a mixture of several to several tens of antibodies) or a polyclonal antibody can also be used as long as CBP can be detected with sufficient specificity. Alternatively, a functional antibody fragment such as Fab, Fab′, F(ab′)2, Fv, scFv, sc(Fv)2, dsFv, or diabody, or a multimer thereof (e.g., a dimer, a trimer, a tetramer, or a polymer) can also be used. The anti-CBP antibody may be a commercially available product.
The detection of the CBP protein can also be carried out by use of mass spectrometry (MS). Particularly, liquid chromatography coupled to mass spectrometer (LC/MS) analysis is sensitive and is therefore advantageous. The mass spectrometry measurement can be carried out, for example, by preparing proteins from the biological sample, labeling the proteins, fractionating the proteins, subjecting the protein fractions to mass spectrometry, and identifying the CBP protein from the mass spectrometry value. An isotope labeling reagent known in the art can be used as the label, and an appropriate labeling reagent can be obtained as a commercially available product. Also, the fractionation can be carried out by a method known in the art and can be carried out using, for example, a commercially available strong cation column.
—Others—
It is known in the art that promoter hypermethylation is partly responsible for reduction in gene expression. Thus, the presence or absence of functional suppression of CBP can be possibly detected with CBP gene promoter methylation as an index. The promoter methylation can be detected by use of a method known in the art, for example, a method which involves directly detecting, by sequencing, change in nucleotide sequence treated with bisulfite having the activity of converting methylated cytosine to uracil, or an indirect detection method using a restriction endonuclease that can recognize (or cleave) a nucleotide sequence before bisulfite treatment but cannot recognize (or cleave) a nucleotide sequence treated with bisulfite.
In this way, when the functional suppression of CBP, for example, loss-of-function inactivating mutation in CBP, has been detected, when the reduction in CBP expression has been detected, or when any of other phenomena that cause inactivation of CBP or reduction in CBP expression (e.g., promoter hypermethylation) has been detected, the patient can be determined as responsive to the treatment of cancer with the compound inhibiting p300 and can be selected as the candidate for the treatment of cancer with the compound inhibiting p300. In this context, the “responsiveness to the treatment of cancer” is an index for deciding whether or not the compound inhibiting p300 can exert therapeutic effects on cancer. The determination of the responsiveness may include not only the determination of the presence or absence of the responsiveness but the evaluation of the degree of the found responsiveness (e.g., evaluation concluding that high responsiveness can be expected or moderate responsiveness can be expected). Thus, according to the degree of functional suppression of CBP, for example, a patient may be selected as a treatment candidate at a level where moderate responsiveness can be expected.
On the other hand, when no functional suppression of CBP has been found, the patient can be excluded from the candidate for the treatment of cancer with the compound inhibiting p300. This can improve the response rate of the treatment.
When p300, which is a target of this treatment, is not normally expressed, there is the risk that the treatment of cancer with the compound inhibiting p300 cannot be effectively carried out. Thus, the normal expression of p300 can also be used as an additional index in the prediction of responsiveness to the treatment of cancer or the selection of a cancer patient. The approach for detecting the expression of p300 is the same as in the detection of CBP expression described above.
<Method for Treating Cancer>
The present invention also provides a method for treating cancer having functional suppression of CBP, comprising using a biological sample derived from a cancer patient, detecting the presence or absence of functional suppression of CBP contained in the biological sample, and administering a compound inhibiting p300 to the patient with the detected functional suppression of CBP.
The “compound inhibiting p300” for use in this treatment is not particularly limited and may be a compound known in the art or may be a compound that is identified by screening mentioned later.
The compound inhibiting p300 can be administered orally or parenterally (e.g., intravenously, intraarterially, or locally) to the cancer patient in various forms such as tablets, powders, granules, capsules, or solutions according to its characteristics. The dose is not particularly limited as long as the amount is effective for treating cancer by inhibiting p300. The dose can be appropriately selected according to the properties of the compound as well as the age, body weight, symptoms, and health conditions of the cancer patient, the severity of the cancer, etc. The frequency of administration is not particularly limited and can be appropriately selected according to a purpose. For example, a daily dose may be administered once a day or may be administered in several portions. In the case of administering the compound inhibiting p300 to a human, the dose ranges from approximately 0.01 mg/kg body weight to approximately 500 mg/kg body weight, preferably from approximately 0.1 mg/kg body weight to approximately 100 mg/kg body weight, per day. In the case of the compound inhibiting p300 to a human, preferably, this compound is administered in one portion or in two to four portions per day, and this administration is preferably repeated at appropriate intervals. Alternatively, the daily dose may exceed the amount described above, if necessary, at a physician's discretion.
This can further inhibit p300 in cancer cells having functional suppression of CBP in the cancer patient and can treat the cancer by the effect of synthetic lethality.
Examples of the cancer targeted by the treatment include, but are not limited to, lung cancer, bladder cancer, lymphoma, and adenoid cystic cancer.
<Reagent for Detecting Presence or Absence of Functional Suppression of CBP>
The present invention also provides a reagent for detecting the presence or absence of functional suppression of CBP in any of the methods described above, the reagent comprising any of the following molecules
(a) to (c) as an active ingredient:
(a) an oligonucleotide primer specifically binding to the CBP gene,
(b) an oligonucleotide probe specifically binding to the CBP gene, and
(c) an antibody specifically binding to the CBP protein.
The polynucleotide primer can be designed on the basis of nucleotide sequence information (e.g., SEQ ID NO: 1 or 2) on the CBP genomic DNA or cDNA such that the primer is suitable for the approach described above or the region to be amplified and such that the formation of an amplification product of a gene other than the CBP gene is prevented as much as possible. Those skilled in the art can carry out such oligonucleotide primer design by a routine method. The length of the oligonucleotide primer is usually 15 to 50 bases long, preferably 15 to 30 bases long, and may be longer than these lengths according to an approach and a purpose.
The polynucleotide probe described above can be designed on the basis of nucleotide sequence information (e.g., SEQ ID NO: 1 or 2) on the CBP genomic DNA or cDNA such that the primer is suitable for the approach described above or the region to be hybridized and such that hybridization to a gene other than the CBP gene is prevented as much as possible. Those skilled in the art can carry out such oligonucleotide probe design by a routine method. The length of the oligonucleotide probe is usually 15 to 200 bases long, preferably 15 to 100 bases long, more preferably 15 to 50 bases long, and may be longer than these lengths according to an approach and a purpose.
Preferably, the oligonucleotide probe is appropriately labeled for use. Examples of the labeling method can include a method which involves labeling the 5′ end of the oligonucleotide by phosphorylation with 32P using T4 polynucleotide kinase, and a method which involves incorporating a substrate base labeled with an isotope (e.g., 32P), a fluorescent dye, or biotin into the oligonucleotide using a DNA polymerase such as Klenow enzyme and a primer such as a random hexamer oligonucleotide (random prime method, etc.).
The oligonucleotide primer and the oligonucleotide probe of the present invention can be prepared using, for example, a commercially available oligonucleotide synthesizer. The oligonucleotide probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like. The oligonucleotide primer and the oligonucleotide probe of the present invention do not have to be consist of natural nucleotides (deoxyribonucleotide (DNA) and/or ribonucleotide (RNA)) and may be composed partially or wholly of non-natural nucleotides. Examples of the non-natural nucleotides include PNA (polyamide nucleic acid), LNA® (locked nucleic acid), ENA® (2′-O,4′-C-ethylene-bridged nucleic acid), and complexes thereof.
The polyclonal antibody serving as the antibody specifically binding to the CBP protein described above can be obtained by immunizing an immunization animal with an antigen (e.g., the CBP protein, a partial peptide thereof, or cells expressing the protein or the partial peptide) and purifying the polyclonal antibody from the antisera of the animal by a conventional approach (e.g., salting-out, centrifugation, dialysis, or column chromatography). The monoclonal antibody serving as this antibody can be prepared by hybridoma method or recombinant DNA method.
Typical examples of the hybridoma method include the method of Kohler and Milstein (Kohler & Milstein, Nature 1975; 256: 495). In this method, antibody-producing cells for use in a cell fusion step are spleen cells, lymph node cells, peripheral leukocytes, or the like of the animal (e.g., mouse, rat, hamster, rabbit, monkey, or goat) immunized with an antigen (e.g., the CBP protein, a partial peptide thereof, or cells expressing the protein or the partial peptide). Antibody-producing cells obtained by the action of the antigen in a medium on these cells or lymphocytes or the like described above isolated in advance from an unimmunized animal may also be used. Various cell lines known in the art can be used as myeloma cells. The antibody-producing cells and the myeloma cells may be derived from different animal species as long as these cells can be fused. Preferably, the antibody-producing cells and the myeloma cells are derived from the same animal species. The hybridomas are produced by, for example, the cells fusion between spleen cells obtained from a mouse immunized with the antigen and mouse myeloma cells and can be obtained by subsequent screening for hybridomas producing the monoclonal antibody specific for the CBP protein. The monoclonal antibody against the CBP protein can be obtained by the culture of the hybridomas or from the ascitic fluid of a mammal that has received the hybridomas.
The recombinant DNA method is an approach which involves cloning DNA encoding the antibody from the hybridomas, B cells, or the like, incorporating this DNA into an appropriate vector, and transferring this vector to host cells (e.g., a mammalian cell line, E. coli, yeast cells, insect cells, or plant cells), followed by the production of the antibody of the present invention as a recombinant antibody (e.g., P. J. Delves, Antibody Production: Essential Techniques, 1997 WILEY, P. Shepherd and C. Dean Monoclonal Antibodies, 2000 OXFORD UNIVERSITY PRESS, and Vandamme A M, et al., Eur. J. Biochem. 1990; 192: 767-775). For the expression of the DNA encoding the antibody, DNA encoding the heavy chain and DNA encoding the light chain may be incorporated into separate expression vectors, which can then be used in the transformation of host cells, or DNA encoding the heavy chain and DNA encoding the light chain may be incorporated into a single expression vector, which can then be used in the transformation of host cells (see WO94/11523). The antibody can be obtained in a substantially pure and homogenous form by culturing the host cells and separating and/or purifying the antibody from the inside or the culture solution of the host cells. The separation and/or purification of the antibody can employ a method for use in usual polypeptide purification. If a transgenic animal (e.g., cattle, goat, sheep, or pig) harboring the antibody gene is prepared by use of a transgenic animal preparation technique, the monoclonal antibody derived from the antibody gene may be obtained in large amounts from the milk of the transgenic animal.
On the basis of the antibody thus obtained or the gene thereof, a functional antibody fragment such as Fab, Fab′, F(ab′)2, Fv, scFv, sc(Fv)2, dsFv, or diabody, or a multimer thereof (e.g., a dimer, a trimer, a tetramer, or a polymer) can be prepared.
In the case of directly detecting the amount of the antibody bound with the CBP protein, the obtained anti-CBP antibody is used either directly or after being labeled with, for example, an enzyme, a radioisotope, a fluorescent dye, or an avidin-biotin system. On the other hand, in the case of carrying out an indirect detection method for detecting the amount of the antibody bound with the CBP protein using a secondary antibody or the like, the obtained anti-CBP antibody (primary antibody) does not have to be labeled. For this detection, a labeled molecule (e.g., secondary antibody or protein A) that recognizes the antibody can be used.
The reagent of the present invention can optionally contain additional components acceptable for reagents, such as sterilized water, saline, a buffer, and a preservative, according to the need, in addition to the molecule described above as an active ingredient.
<Method for Screening for Compound for Use in Treatment of Cancer>
The present invention also provides a method for screening for a compound for use in the treatment of cancer having functional suppression of CBP, the method comprising the step of selecting the compound by using whether or not to inhibit p300 as an index.
Examples of the test compound that is applied to the screening system of the present invention include, but are not particularly limited to, synthetic low-molecular compound libraries, expression products of gene libraries, peptide libraries, siRNA, antibodies, substances released by bacteria, extracts and culture supernatants of cells (microbes, plant cells, or animal cells), purified or partially purified polypeptides, marine organism-, plant-, or animal-derived extracts, and random phage peptide display libraries. The test compound may be a derivative of a p300 inhibitor (e.g., a C646 derivative) known in the art.
In the present invention, the “inhibition of p300” includes both of inhibition of p300 activity and inhibition of p300 expression. Since the loss of the histone acetyltransferase activity of p300 is considered to contribute to the synthetic lethality of CBP and p300, the inhibition of p300 used as an index of screening is preferably inhibition of the histone acetyltransferase activity of p300. For example, a detection method using a radioisotope (Lau O D, et al., J. Biol. Chem. 2000; 275: 21953-21959), a method which involves fluorescently detecting CoA-SH generated as by-products during histone acetyltransferase reaction (Gao T, et al., Methods Mol Biol. 2013; 981: 229-38), and a detection method using NADH (Berndsen C E, Denu J M. Methods. 2005; 36: 321-331) can be used for detecting the histone acetyltransferase activity.
In the screening, the test compound can be allowed to act on this detection system, followed by the detection of histone acetyltransferase activity. As a result of the detection, when the activity is decreased as compared with the histone acetyltransferase activity of p300 in a control (e.g., without the addition of the test compound), the activity of p300 can be evaluated as being inhibited.
As CBP and p300 are in a paralogous relationship, the compound obtained by screening is preferably more specific for p300 from the viewpoint of reduction in adverse reactions, etc. Whether or not to be specific for p300 can be evaluated by, for example, a binding experiment or an activity inhibition experiment on each molecule. Thus, the screening of the present invention may comprise the step of selecting the compound by using whether or not to be more specific for p300 as an index.
In the case of detecting the inhibition of p300 expression, for example, the test compound can be allowed to act on cells expressing p300, followed by the detection of the p300 expression at the transcriptional level or translational level by the method described above. Alternatively, a reporter assay system based on an expression construct containing a reporter gene linked downstream of a p300 promoter may be used. As a result of the detection, when the expression is decreased as compared with the expression of p300 (or the expression of the reporter as a substitute therefor in the reporter system) in a control (e.g., without the addition of the test compound), the expression of p300 can be evaluated as being inhibited.
The compound identified by the screening of the present invention can be mixed with a pharmacologically acceptable carrier and formulated by a pharmaceutical method known in the art to prepare a pharmaceutical product. Examples of the pharmacologically acceptable carrier include, but are not limited to, sterilized water, saline, plant oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, fragrances, excipients, vehicles, antiseptics, binders, diluents, tonicity agents, soothing agents, expanders, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, corrigents, solubilizers, and other additives.
Hereinafter, the present invention will be described further specifically with reference to Examples. However, the present invention is not intended to be limited by these Examples.
1. Material and Method
(1) Cell Line
A549, H1299, H157, SQ5, H1703, LK2, H520 (NSCLC), RL, Loucy, RC-K8, U2932, Ramos, Farage, SUP-T1, WSU-NHL, VAL, SUDHL5, Jurkat, TE8, and TE10 were separately cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS). MRC-5 cells (normal fibroblasts), HEK293T cells (immortalized renal epithelial cells), and RPE-1 cells (immortalized retinal epithelial cells) were separately cultured in DMEM supplemented with 10% FBS.
(2) Short Interfering RNA (siRNA)
ON-TARGET plus SMARTpool siRNA (GE Healthcare Dharmacon Inc.) was used in knockdown of various proteins. Lipofectamine R NAiMAX (Invitrogen Corp.) was used in transfection. Non-targeting siRNA (L-001810-10) was used as a negative control.
(3) Immunoblot Analysis Immunoblotting was performed as described in the literature (Ogiwara H, et al., Oncogene 2011; 5; 30: 2135-46) using antibodies specific for the following proteins: CBP (Santa Cruz Biotechnology, Inc.; sc-369X), p300 (Santa Cruz Biotechnology, Inc.; sc-48343X), H3 (Active Motif; 39163), H3K18ac (Millipore Corp.; 07-354), β-actin (Cell Signaling Technology, Inc.; 4970), cleaved PARP (Cell Signaling Technology, Inc.; 5625), p21/CDKN1A (Cell Signaling Technology, Inc.; 2947), and LC3B (Cell Signaling Technology, Inc.; 3868).
(4) Cell Survival Assay
The effect of siRNA knockdown on the survival of cancer cells was evaluated using clonogenic survival assay. Each cancer cell line was transfected with siRNA (50 nM) using Lipofectamine RNAiMAX (Invitrogen Corp.). Two days later, the cells were trypsinized, counted, reseeded in specified numbers in 6-well culture dishes, and further cultured for 12 days (or for 14 days for knockdown of various HATs) to allow colony formation. The cells were then fixed for 5 minutes in a solution containing 50% (v/v) methanol/0.01% (w/v) Crystal Violet, and the number of colonies was counted.
The viability was determined by examining intracellular ATP level using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega Corp.). Each cancer cell line was transfected with siRNA (50 nM) using Lipofectamine RNAiMAX (Invitrogen Corp.). Two days later, the cells were trypsinized, counted, and reseeded in specified numbers in 96-well plate. In order to measure the cell viability, CellTiter-Glo Luminescent Cell Viability Assay kit (Promega Corp.) was added to the cells, and fluorescence was measured using Envision (PerkinElmer, Inc.).
(5) Cell-Cycle Analysis
Cells were trypsinized, centrifuged, washed with PBS, and fixed in 70% ice-cold ethanol. The cells were then centrifuged again and incubated in PBS containing 200 μg/ml RNase A and 5 μg/ml propidium iodide. The cell-cycle distribution was analyzed by Guava flow cytometry (Millipore Corp.).
(6) Apoptosis Analysis by Annexin V/PI Staining
In order to detect apoptotic cells by flow cytometry, Annexin V-FITC/PI apoptosis detection kit (F. Hoffmann-La Roche, Ltd.) was used according to the manufacturer's instruction manual. Briefly, the cell pellet was suspended in a 1× binding buffer and incubated with Annexin V-FITC and PI for 20 minutes in the dark. Subsequently, the fluorescence of the cells was analyzed by flow cytometry.
(7) Production of shRNA Lentivirus In order to prepare tet-inducible cell lines, each cell line was transduced with shRNA-expressing lentivirus vector-derived pTRIPZ (Open Biosystems). 293T cells were cotransfected with shRNA-encoding plasmids and packaging plasmids using Trans-Lentiviral™ Packaging System (Open Biosystems). On the next day, the medium was replaced with a fresh growth medium, and the supernatant containing the lentivirus was recovered and concentrated by centrifugation.
(8) In Vivo Analysis
The numbers of tet-inducible cell lines LK2-shp300 cells and LK2-shNT cells (2×106 cells/mouse in 50% Matrigel; BD Biosciences) were counted, and the cells of each line were re-suspended in a 1:1 mixture of a medium and Matrigel (BD Biosciences) on ice. The cells were subcutaneously injected into the flanks of 7-week-old female BALB/c-nu/nu mice (CLEA Japan, Inc.) using a protocol approved by the Ethical Committee on Animal Experiments at the National Cancer Center. Three weeks later when tumor size reached 200 mm3 or larger, the mice were randomly divided into 2 groups and fed with either a diet containing doxycycline (200 ppm) or a control diet. Tumor growth was measured twice a week using a caliper. The volume of the transplanted tumor was calculated every 3 to 4 days using a caliper according to the following formula: V=L×W2/2 wherein V represents volume (mm3), L represents the largest diameter (mm), and W represents the smallest diameter (mm). At the end of the experiment, the mice were sacrificed according to standard protocols.
(9) Statistical analysis All experiments were performed in triplicate. Data are shown as the mean±SD. The differences between drug-treated cells and untreated cells were evaluated using Student's t-test. Statistically significant differences are indicated by asterisks (“*”, P<0.05; “**”, P<0.01; “***”, P<0.001; “****”, P<0.0001).
2. Results
(1) p300-Dependent Growth of CBP-Mutated Cancer Cells
We compared the effect of siRNA-mediated p300 depletion on growth of CBP-proficient and CBP-mutated cancer cell lines, using a cell growth assay and clonogenic survival assay (
p300 knockdown did not affect the growth of the non-cancerous fibroblast line MRCS, the immortalized renal epithelial cell line HEK293T, and the immortalized retinal epithelial cells RPE-1 expressing CBP and p300 (
These results suggested that CBP-mutated cancer cells depend on p300 for growth.
(2) Sensitivity of CBP-Mutated Lung Cancer and Lymphoma to p300 Inhibitor C646
We next examined the influence of a p300 inhibitor C646 on CBP-mutated lung cancer cells and lymphoma cells. As a result, higher sensitivity of the CBP-mutated cancer cells to C646 than that of the CBP-proficient cancer cells was observed (
(3) Induction of Apoptosis by p300 Depletion in CBP-Mutated Cancer Cell
We next examined the mechanism underlying inhibition by p300 depletion or inhibition in CBP-mutated cancer cells. siRNA-mediated depletion of p300 in CBP-mutated H1703 cells increased the amount of cleaved PARP (biomarker of apoptosis), but did not increase the amount of p21/CDKN1A (biomarker of cell senescence) or LC3B (biomarker of autophagy) (
In consideration of the results of using the p300 inhibitor C646 described above (
(4) In Vivo p300-Dependent Growth of CBP-Mutated Cancer Cells
CBP-mutated LK2 cells were used as in vivo preclinical validation models. Cells expressing non-targeting shRNA or shp300 were prepared using a tetracycline-inducible shRNA expression system and subcutaneously transplanted to nude mice. After transplanted tumor size reached 200 mm3 or larger, doxycycline was administered to the mice to induce RNAi. Tumor growth was measured over time. As shown in
(5) Influence of Knockdown of Various HATs on Cell Survival Rate
The influence of knockdown of various HATs in A549 cells (CBP-proficient) and H520 cells (CBP-mutated) on their cell survival rates was observed. The influence of knockdown of various HATs on the cell survival rate was not observed in the A549 cells, whereas the knockdown of p300 in the H520 cells was confirmed to significantly reduce the cell survival rate (
(6) Influence of Knockdown of p300 on c-Myc Expression
Change in c-Myc protein expression caused by p300 knockdown in various cells was observed.
Change in c-Myc protein expression by p300 knockdown was not observed in CBP-proficient H1299 cells, SQ5 cells, and H157 cells, whereas the p300 knockdown was confirmed to significantly decrease c-Myc protein expression in CBP-mutated LK2 cells, H1703 cells, H520 cells, TE8 cells, and TE10 cells, and CBP-knockout H1299 cells (
Reduction in c-Myc expression was observed in the cells having functional suppression of both CBP and p300, indicating that c-Myc may be used as a marker for confirming the effect of a p300 inhibitor on cells found to have functional suppression of CBP.
The present invention provides a therapeutic strategy for specifically targeting cancer cells having functional suppression of CBP. In the present invention, it has been found that CBP and p300 are in the relationship of synthetic lethality, and treatment inhibiting p300 is a promising approach for the treatment of cancer having functional suppression of CBP. It has also been revealed that this therapeutic strategy achieves efficient treatment based on companion diagnostics because a p300 inhibitor can be administered to a cancer patient selected with functional suppression of CBP as an index. Thus, the present invention can have a great contribution to the medical field, particularly, the field of cancer treatment.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-032928 | Feb 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/054991 | 2/23/2015 | WO | 00 |
