WO2010013633A1 - Methods for prediction of rb status and sensitivity to plk1 inhibitor of cell - Google Patents

Abstract

Disclosed is a method for predicting the sensitivity of a cancer cell to a PLK1 inhibitor.  Also disclosed is a method for predicting the RB status of a cancer cell. The sensitivity to a PLK1 inhibitor or the RB status can be predicted through the following steps: determining the expression level of cyclin D1 gene and the expression level of p16 gene in a biological sample collected from a patient; determining the ratio of the expression level of cyclin D1 gene to the expression level of p16 gene; and predicting that the PLK1 inhibitor is effective or the RB function is abnormal when the ratio of the expression levels is smaller compared with that in a control cell.

Description

Method for predicting RB status of cells and sensitivity to PLK1 inhibitor

The present invention relates to a method for predicting the sensitivity of cancer cells to a PLK1 inhibitor and a method for predicting RB status.

With the development of tailor-made treatment according to the genetic characteristics of individual cancers, clinical diagnostic techniques are becoming recognized as important. For example, even when the same drug is used, its therapeutic effect and side effects (toxicity) vary from patient to patient. Therefore, it is desirable to administer drugs with high therapeutic effects and no or no side effects for each patient. In particular, anticancer drugs have therapeutic effects that vary greatly depending on the type of various cancer cells, and often have side effects. Predict drug efficacy and side effects before administration, and provide the most appropriate treatment for each patient. However, it is desirable for enhancing the therapeutic effect.

It is known that various pathways such as Hedgehog pathway, Wnt pathway, apoptosis pathway via Survivin gene, Ras pathway, p53 pathway and RB pathway are involved in canceration. In order to predict the efficacy of anticancer drugs, it is desirable to determine which pathway abnormalities are caused by the patient's cancer and to administer appropriate anticancer drugs. It is rare.

RB protein that forms the core of the RB pathway is one of tumor suppressor genes and plays an extremely important role in the negative control of the cell cycle. The RB protein is involved in a G1 checkpoint that blocks the transition from G1 to S phase. The RB protein is derived from the G1 phase by directly binding to the transactivation domain of E2F and by forming a complex with E2F and binding to the promoter of a gene required for the transition from G1 phase to S phase. Suppresses gene transcription required for transition to S phase. When the RB function is impaired, the cell cycle becomes abnormal and contributes to canceration.

¡Chromosomal separation without error during cell division is required to maintain correct ploidy and genomic integrity. Errors during cell division are speculated to cause aneuploidy and cancer. In order for daughter cells to inherit chromosomes correctly, it is important that the chromosomes are evenly separated and that cytokinesis occurs between the two sets of separated chromosomes. It is known that a kinase called PLK1 (Polo-likeＫkinase 1; polo-like kinase 1) is involved in these processes in cell division.

PLK1 is a phosphorylating enzyme that is activated during cell division. Increased expression of PLK1 has been observed in various human cancers (malignant tumors), and this increased expression is known to be associated with poor prognosis in various types of cancer. Therefore, it has been proposed to use PLK1 as a disease marker. Furthermore, it has been confirmed that when PLK1 is specifically deficient in cancer cells, the viable cell rate decreases due to apoptosis. Therefore, PLK1 inhibitors are being developed as promising candidates for the development of anticancer agents.

Therefore, an object of the present invention is to develop a method for predicting the effect of a PLK1 inhibitor as a cancer therapeutic agent. Another object of the present invention is to identify a gene that is expressed in correlation with the RB function in order to investigate the relationship between the RB function of the cell and the effect of the PLK1 inhibitor.

The present inventor analyzed a gene expression pattern related to RB function, and identified a gene expressed in correlation with RB function. The present inventor has found that the cyclin D1 gene and the p16 gene are particularly effective for predicting whether or not the RB function of the cell is abnormal because the correlation with the RB function is high. Furthermore, the present inventor has confirmed that cancer cells with abnormal RB function are highly sensitive to a PLK1 inhibitor, and that cell death is induced by administration of the PLK1 inhibitor. Therefore, the present invention can predict whether or not the RB function is normal based on the expression profile of the gene expressed in correlation with the RB function, in particular, the expression ratio of the cyclin D1 gene and the p16 gene, and further inhibits PLK1 inhibition. It has been clarified that the effect of the agent can be predicted, and has been completed.

That is, the present invention includes the steps of determining the expression level of the cyclin D1 gene and the expression level of the p16 gene in the patient-derived biological sample, determining the ratio of the expression level of the cyclin D1 gene to the expression level of the p16 gene, And predicting that the PLK1 inhibitor is effective when the ratio of the expression levels is small compared to the control cells.
The present invention also includes the steps of determining the expression level of the cyclin D1 gene and the expression level of the p16 gene in the patient-derived biological sample, determining the ratio of the expression level of the cyclin D1 gene to the expression level of the p16 gene, Predicting whether the RB function in the cell is abnormal, including predicting that the RB function in the cell in the biological sample is abnormal when the ratio of expression levels is small compared to the control cell Regarding the method.
In the method, the ratio of expression levels is represented by the expression ratio of cyclin D1 / p16, and the value of the expression ratio of cyclin D1 / p16 is expressed by the following formula:

Can be represented by

In another aspect, the present invention relates to whether or not RB function in cells in a biological sample is abnormal based on RB gene mutation, RB gene expression level and / or RB protein production level in a biological sample derived from a patient. The present invention relates to a method for predicting the effect of a PLK1 inhibitor, comprising the steps of predicting and predicting that the PLK1 inhibitor is effective when the RB function is abnormal.
Furthermore, the present invention determines the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a biological sample derived from a patient, and FIG. The PLK1 inhibitor is effective when the expression level of the gene described in 1 is smaller than that of the control cells and / or when the expression level of the gene described in FIG. 8 is higher than that of the control cells. The method of predicting the effect of a PLK1 inhibitor.

The present invention also includes the step of determining the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a biological sample derived from a patient, When the expression level of the described gene is small compared to the control cell and / or when the expression level of the gene described in FIG. 8 is large compared to the control cell, the RB function in the cell is abnormal. A method for predicting whether or not RB function in a cell is abnormal.
In the above method, the expression level of one or more genes described in FIGS. 7 and 8 is determined, the gene described in FIG. 7 includes the cyclin D1 gene, and the gene described in FIG. 8 includes the p16 gene. Is desirable.

The patient-derived biological sample can contain cancer cells, preferably contains lung cancer cells, and more preferably contains small cell cancer cells.
In another aspect, the present invention provides a pharmaceutical composition for treating a cancer patient having cancer cells with abnormal RB function, the pharmaceutical composition comprising a pharmaceutically effective amount of a PLK1 inhibitor. In addition, the present invention also relates to a drug for inducing cell death in cells having abnormal RB function, the drug comprising a pharmacologically effective amount of a PLK1 inhibitor.
The pharmaceutical composition and drug can be administered to cancer cells predicted to have abnormal RB function by the method of the present invention, or to cells predicted to have abnormal RB function.

As yet another aspect, the present invention determines a cyclin D1 gene expression level and a p16 gene expression level in a patient-derived biological sample, and determines a ratio of the cyclin D1 gene expression level to the p16 gene expression level. And a step of administering a PLK1 inhibitor to a patient when the ratio of expression levels is smaller than that of control cells.
In the above treatment method, the ratio of expression levels can be expressed by the expression ratio of cyclin D1 / p16, and the value of the expression ratio of cyclin D1 / p16 is expressed by the following formula:

Can be represented by

In yet another aspect, the present invention relates to whether the RB function in cells in the biological sample is abnormal based on RB gene mutation, RB gene expression level and / or RB protein production level in a biological sample derived from a patient. The present invention relates to a method for treating cancer, comprising: a step of predicting whether or not, and a step of administering a PLK1 inhibitor to a patient when the RB function is abnormal.
The present invention also includes the step of determining the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a biological sample derived from a patient, A step of administering a PLK1 inhibitor to a patient when the expression level of the described gene is small compared to the control cell and / or when the expression level of the gene described in FIG. 8 is large compared to the control cell And a method for treating cancer.
In the above treatment method, the expression level of one or more genes described in FIGS. 7 and 8 is determined, the gene described in FIG. 7 includes the cyclin D1 gene, and the gene illustrated in FIG. 8 includes the p16 gene. It is desirable.
The present invention relates to one or more isolated nucleic acid sequences selected from the group consisting of FIG. 7 and FIG. 8 that are associated with the function of RB, and also comprising nucleic acids that bind to said one or more nucleic acid sequences. It also relates to an array containing.

The present invention provides an expression profile of a gene whose expression increases or decreases in relation to RB function, and more particularly based on the expression ratio of cyclin D1 gene and p16 gene than that of cyclin D1 gene alone or p16 gene alone. It is possible to predict whether or not the RB function is abnormal with high accuracy. Further, according to the present invention, sensitivity to a PLK1 inhibitor can be predicted based on the expression ratio of the cyclin D1 gene and the p16 gene. For example, before administering a PLK1 inhibitor, a biological sample is collected from a cancer patient, and based on the expression ratio of the cyclin D1 gene and the p16 gene, whether the RB function is normal and sensitivity to the PLK1 inhibitor And the present invention can be administered to a patient who is highly sensitive to a PLK1 inhibitor, that is, a drug containing the PLK1 inhibitor is administered only to a patient who is expected to be effective in administering the PLK1 inhibitor. It helps when selecting an anticancer drug to be administered to a patient.

It is a figure which shows the clustering of 30 cell lines by RB function signature 194 gene. It is a graph which shows the relationship between the gene expression level ratio of cyclin D1 and p16, and RB status. It is a figure which shows that PLK1 inhibitor induces apoptosis in an RB abnormality-dependent manner with respect to U-2UOS RB matched pair cell lines. It is a figure which shows what kind of pathway RB controls the activity of an E2F transcription factor. It is a figure which shows that a cyclin D1 and p16 gene expression ratio can identify actual RB status. It is the figure which showed that the sensitivity of a PLK1 inhibitor correlates with the RB status estimated from the gene expression ratio of cyclin D1 and p16. A gene whose expression level decreases when RB function is abnormal is shown. A gene whose expression level increases when RB function is abnormal is shown.

As used herein, a patient refers to a human who has or may have cancer, but besides humans, veterinary uses, i.e., non-human primates, Mammals such as dogs, cats, cows and horses can be targeted.

Examples of biological samples include body fluids obtained from patients, body fluids containing cells such as blood, sputum, and intraperitoneal washings. The body tissue obtained from the patient is preferably a cancer tissue. Moreover, not only a patient's cell but the primary culture or the subcultured thing may be used.

The RB function state is also referred to as “RB status” in this specification. The RB function refers to whether or not the RB protein functions normally. The case where the RB function is abnormal includes not only the abnormality of the RB protein itself but also the case where the function of the RB protein is inhibited by a viral protein and is not functioning normally. However, it does not include the case where the RB protein is not functioning normally due to abnormal protein function upstream of the RB protein in the RB pathway.

Either absolute or relative amount of gene expression may be measured as the gene expression level. For example, the expression levels of cyclin D1 gene and p16 gene are as follows: 1) cyclin D1 mRNA and p16 mRNA are detected 2) cyclin D1 protein and p16 protein are detected 3) cyclin D1 and p16 protein biological activities Can be determined by any one or more of detecting. For example, gene amplification methods such as real-time PCR and RT-PCR, microarrays, Northern hybridization, and immunological measurement methods such as IHC, ELISA, and Western blotting can be used, but are not particularly limited. The expression level can also be measured by hybridization of a target gene and a complementary probe using cells or tissues.

The expression ratio between the cyclin D1 gene and the p16 gene in the cell whose RB function or PLK1 inhibitor sensitivity is to be predicted can be determined as the ratio of the expression level of the cyclin D1 gene and the p16 gene in the control cell as follows: .

At this time, when the expression ratio of cyclin D1 / p16 is large, it can be predicted that the RB function is normal and the sensitivity of the PLK1 inhibitor is low. On the other hand, when the expression ratio of cyclin D1 / p16 is small, the RB function is abnormal, and the sensitivity of the PLK1 inhibitor is high, that is, cell death can be effectively induced by administration of the PLK1 inhibitor. it can.

Furthermore, log ₁₀ (expression ratio of cyclin D1 / p16) can also be used to predict RB function and PLK1 inhibitor sensitivity. That is, when log ₁₀ (the expression ratio of cyclin D1 / p16) is large, it can be predicted that the RB function is normal and the sensitivity of the PLK1 inhibitor is low. On the other hand, when log ₁₀ (the expression ratio of cyclin D1 / p16) is small, the RB function is abnormal and the sensitivity of the PLK1 inhibitor is high, that is, cell death is effectively caused by administration of the PLK1 inhibitor. It can be predicted that it can be guided.

Whether the RB function is abnormal and whether the sensitivity of the PLK1 inhibitor is high or not, the value of the expression ratio of cyclin / p16 and the log ₁₀ (expression ratio of cyclin D1 / p16), which are the criteria for judgment ) Value varies depending on the control cell X. This value can be easily set by those skilled in the art by measuring the expression levels of cyclin and p16 in several cell lines with known RB functions and the target cell X. For example, when HeLa S3 is used as the cell X, when log ₁₀ (the expression ratio of cyclin D1 / p16) is larger than 0.6, the RB function is normal and the sensitivity of the PLK1 inhibitor is low. Can be predicted to be low. On the other hand, when log ₁₀ (the expression ratio of cyclin D1 / p16) is smaller than 0.6, the RB function is abnormal, and the sensitivity of the PLK1 inhibitor is high, that is, cell death by administration of the PLK1 inhibitor Can be effectively induced.

Further, based on the expression profile of one or more genes described in FIG. 7 and / or one or more genes described in FIG. 8 in a biological sample derived from a patient, RB status of the patient's cancer cells, and PLK1 Sensitivity to inhibitors can be predicted. Specifically, when the expression level of the gene described in FIG. 7 in the biological sample is small compared to the control cells, or when the expression level of the gene described in FIG. 8 is large compared to the control cells, It can be predicted that the RB function is abnormal and the sensitivity to the PLK1 inhibitor is high. In this method, it is desirable to compare both the expression level of the gene described in FIG. 7 and the expression level of the gene described in FIG. 8 with the control cells. As the control cell, a cell confirmed to have normal RB function can be used. The above prediction can be made based on the expression level of one or more genes shown in FIG. 7 and / or one or more genes shown in FIG. 8, but preferably 5 or more of each of the genes described in FIG. More preferably, the above prediction can be made based on 10 or more genes, more preferably 50 or more genes. In addition, the one or more genes described in FIG. 7 preferably include the cyclin D1 gene, and the one or more genes described in FIG. 8 desirably include the p16 gene.

It is desirable to use a biological sample derived from a patient containing or suspected of containing cancer cells. The cancer referred to in this specification is not particularly limited, and may infiltrate (infiltrate) or metastasize to a boundary with another tissue such as a malignant tumor or a malignant neoplasm, and may increase in various parts of the body. Tumors that threaten the life of the host and include carcinomas derived from epithelial tissues (cancer), sarcomas derived from connective and mesenchymal tissues, leukemias, and malignant lymphomas. For example, the method of the present invention can be applied to lung cancer cells, particularly small cell lung cancer cells.

The gene expression profile indicates information such as the expression pattern of a gene in a specific cell under a certain condition, the presence or absence of expression of a known and unknown gene, the expression level of all the genes to be expressed, and the like. In order to comprehensively analyze the genes described in FIG. 7 and FIG. 8, in addition to nucleic acid amplification methods such as RT-PCR and real-time PCR, microarray, Northern hybridization, differential display, differential hybridization, subtraction, etc. Any analysis method known to those skilled in the art can be used.

It has been clarified by the present inventor that cancer cells having an abnormal RB function are highly sensitive to a PLK1 inhibitor and can selectively cause cell death in cells having an abnormal RB function. Therefore, a patient having cancer cells predicted to have abnormal RB function by the above method or other methods is selected and a pharmaceutical composition containing a pharmacologically effective amount of a PLK1 inhibitor is administered. It is effective to do. On the other hand, even if a PLK1 inhibitor is administered to a patient having cancer cells having no abnormality in RB function, only side effects appear and the possibility that the effect cannot be expected is high. Therefore, according to the present invention, an appropriate treatment method can be selected after predicting the RB function in a cancer cell of a patient in advance.

Based on this finding, by specifying a patient group to be administered to cancer patients having cancer cells with abnormal RB function, a pharmaceutical composition containing a PLK1 inhibitor can be effectively administered. . A pharmaceutical composition comprising a PLK1 inhibitor typically comprises pharmacologically acceptable excipients and / or other additives. A pharmacologically effective amount of a PLK1 inhibitor against cancer is included in the pharmaceutical composition.

The administration method of the pharmaceutical composition of the present invention includes oral administration such as buccal and sublingual, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, parenteral administration such as pulmonary administration, etc. It can be appropriately changed according to the cancer site and symptoms of the patient. Therefore, for oral administration, tablets, pills, capsules, powders, granules, extracts, liquids, etc., and for parenteral administration, injections, suppositories, eye drops, nasal drops , Ointments, liquids, patches, nasal agents, inhalants, sprays, and the like.

Preferably, it can be formulated together with pharmacologically acceptable excipients and / or additives by a number of methods well known to those skilled in the art. Examples of excipients include, but are not limited to, lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinyl pyrrolidone, magnesium aluminate metasilicate, and the like. Examples of other additives include excipients, stabilizers, preservatives, buffering agents, flavoring agents, suspending agents, emulsifiers, flavoring agents, solubilizing agents, coloring agents, and thickening agents. Moreover, it is good also as a liquid agent using aseptic purified water, especially distilled water for injection, physiological saline, alcohol (for example, ethanol), vegetable oil, glucose aqueous solution, etc. Ointments include not only oleaginous bases (eg, petrolatum, paraffin, etc.), but also creams, water-soluble bases (eg, polyethylene glycol bases), suspension bases (e.g., petroleum jelly, paraffin, etc.) For example, those using cellulose) are included.

A person skilled in the art can arbitrarily change the dose of the pharmaceutical composition containing the PLK1 inhibitor according to the type of the PLK1 inhibitor, the patient's medical condition, the location of the lesion, age, weight, sex, administration route and the like. As an example, the pharmaceutical composition includes 0.1 to 1000 mg / day of a PLK1 inhibitor per unit dose, depending on the route of administration, for example, 1 mg to 600 mg / day of a PLK1 inhibitor. The pharmaceutical composition of the present invention may be further combined with other anticancer agents or anticancer treatments.
In addition, a drug containing a PLK1 inhibitor can be used to induce cell death in cells with abnormal RB function. Cell death includes apoptosis, necrosis, autophagy and the like.

Any PLK1 inhibitor can be used as the PLK1 inhibitor, and Compound Example 38 and Example 186 described in WO2006 / 049339, Compound Example 5 and Example 81 described in WO2008 / 081914, BI 2536 (for example, Curr Biol. 2007 2007 Feb 20; 17 (4): 304-15), ON01910 (Cancer Cell. 2005 2005 May; 7 (5): 497), GSK461364, ZK-Thiazolidinone (Mol Biol Cell. 2007 2007 Oct; 18 (10): 4024-36), Cyclapolin 1 (Nat. Chem. Biol. 2006, Nov .; 2, (11): 608-617), but not limited thereto. The structural formulas of Compound Example 38 and Example 186 described in WO2006 / 049339 and Compound Example 5 and Example 81 described in WO2008 / 081914 are shown below.

In addition, the treatment method and pharmaceutical composition of the present invention can be used in combination with other known anticancer agents or in combination with other known anticancer treatment methods. Examples of other known anticancer agents include anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, plant-derived anticancer agents, and anticancer platinum coordination. Examples include, but are not limited to, compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, monoclonal antibodies, interferons, and biological response modifiers. Other known anti-cancer treatments can include, but are not limited to, radiation therapy.

The inventor has identified 194 genes related to RB function. Among them, 90 genes described in FIG. 7 decrease in expression when RB function is abnormal, and 104 genes described in FIG. 8 increase in expression when RB function is abnormal. It has been confirmed that there is a tendency. By analyzing the expression profiles of the genes described in FIGS. 7 and 8, it is possible to predict more accurately whether or not the RB function is abnormal. By using an array containing nucleic acids that bind to the nucleic acid sequences of the genes described in FIGS. 7 and 8, the expression profile can be analyzed. Such arrays can be made based on known methods. For example, based on the method described in ExpressionExpressprofiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer, Hughes, TR; Mao, M; Jones, AR, et al. NATURE BIOTECHNOLOGY 19, 342-347 (2001) Can do.

According to the present invention, by predicting whether or not the RB function is abnormal, it is possible to predict the sensitivity to the PLK1 inhibitor, that is, whether or not the PLK1 inhibitor is effective. As described above, the RB function is predicted based on the expression ratio of cyclin D1 / p16 or log ₁₀ (expression ratio of cyclin D1 / p16), or the expression level of the genes described in FIG. 7 and FIG. It is possible to determine based on the presence / absence of mutation of the RB gene, the expression level of the RB gene and / or the production amount of the RB protein. Moreover, since RB function is known to be inactivated by viral infection such as HPV, it can be predicted by examining infection with HPV.

It is known to those skilled in the art to measure the presence / absence of mutation of the RB gene, the expression level of the RB gene and / or the production amount of the RB protein. The RB gene mutation includes not only a mutation in the region encoding the RB gene but also a mutation in the promoter region. The presence or absence of mutations in the RB gene is determined by sequencing the RB gene, Southern blotting, Northern blotting, methods based on gene amplification such as PCR-SSCP and ARMS, ASO, Taqman-PCR, Invader, and DNA chip And can be determined by any method known to those skilled in the art. The amount of RB gene expression and the amount of RB protein produced can be measured by detecting the expression and loss of RB gene mRNA, detecting the RB protein, and detecting the biological activity of the RB protein. The expression amount of RB gene and the production amount of RB protein are, for example, gene amplification methods such as real-time PCR and RT-PCR, microarray, Northern hybridization and immunological measurement methods such as IHC, ELISA and Western blotting, RB protein It can be measured by any method known to those skilled in the art, such as a functional assay.

Thus, whether or not the RB function of the cell is abnormal is predicted, and when the RB function is predicted to be abnormal, it is determined that the cell is highly sensitive to the PLK1 inhibitor, and PLK1 Inhibitors are judged to be effective. Moreover, when it is estimated that the RB function of a cell is abnormal, it can be judged that administration of a PLK1 inhibitor is effective for a patient from which the cell is derived. That is, there is provided a cancer treatment method in which the RB function of cells contained in a biological sample of a patient is predicted, and the PLK1 inhibitor is administered when the RB function is abnormal.

<Determination of RB status>
The following 30 human cancer cell lines were used to determine RB status.
T24, U-2 OS, SF-268, DLD-1, HCT 116, Hep G2, NCI-H460, A549, UACC-62, MDAH-2774, PANC-1, SU.86.86, NCI-N87, Hs 746T, KATO III, SCC-25, J82, HeLa S3, Du 145, TCCSUP, MDA-MB-436, MDA-MB-468, SF-539, HeLa, C-33 A, Lu-135, NCI-H128, NCI- H1417, NCI-H69, NCI-H596

Among the above 30 cell lines, information on RB protein function inhibition by RB mutation, expression suppression, or viral protein expression was obtained from 11 literature cell lines (Table 1). These cell lines were used for analysis as RB dysfunction because RB protein is considered to be nonfunctional.

For the remaining 19, RB status was determined by RB protein functional assay.
The RB protein function assay is a method for examining the status of intracellular RB via the inhibitory effect of an exogenous E2F transcriptional reporter. RB is a central molecule that regulates the activity of the E2F transcription factor in the cell, and its regulatory mechanism is as shown in FIG. RB suppresses the transcriptional activity of E2F, but this inhibitory effect is lost when RB is phosphorylated by Cdk4 / cyclin D. Furthermore, Cdk4 / cyclin D phosphorylase activity is inhibited by its intracellular inhibitory protein, p16. For this reason, the status of intracellular RBs can be examined by exogenously introducing p16 and E2F transcription reporters into cells. When RB status is examined by this method, cells whose E2F transcription reporter activity is inhibited by the introduced p16 are identified as normal RB function, and cells whose E2F transcription reporter activity does not change even when p16 is introduced are identified as abnormal RB function. Can do.
By RB protein functional assay, it was determined that 16 were normal and 3 were abnormal as shown in Table 2.

As a result, it was confirmed that of the 30 cell lines used, 16 cell lines had normal RB function and 14 cell lines had abnormal RB function.

<Extraction of genes that correlate with RB function>
In order to extract genes that correlate with the presence or absence of RB protein function, the gene expression of 16 cell lines with normal RB function and 14 cell lines with abnormal RB function was measured using a microarray. Pearson correlation coefficient was calculated between the expression level at 1 and the state of RB function (normal is 1 and abnormality is 0).
Each cell line was cultured under normal conditions, and a total RNA prepared from the cells after a certain period of time was used for gene expression. For the purification of total RNA, RNeasy kit (QIAGEN) was used, and human 3.0 A1 (custom array made by Agilent Technologies) was used as a microarray, and labeling reaction and hybridization were performed according to the protocol of Agilent. Since Agilent's system is a method of competitively hybridizing two samples labeled with different dyes on one array, Human Universal Reference RNAs (HURR) (Stratagene) is commonly used as a reference RNA. It was used so that the expression level between cell lines could be compared with each other.

The fluorescence signal read by the scanner was analyzed with Resolver® (Rosetta® Biosoftware), and the expression level ratio was calculated. For analysis of gene expression data, Resolver, R (GNU freeware), and MATLABMAT (MathWorks) were used.

Was used to determine the expression level ratio.

As a result, a gene corresponding to a probe satisfying the following conditions was identified as a gene that correlates with “RB function signature”, that is, presence or absence of RB protein function.
(1) The standard deviation of the expression log ratio (bottom 10) in all 30 samples is 0.1 or more.
(2) The p-value of the Pearson correlation coefficient between the expression log ratio (bottom 10) and the RB functional state in all 30 samples is 0.001 or less.
The selected “RB function signature” is shown in Table 3 below.

Clustering of 30 cell lines in which the RB function was confirmed to be normal or abnormal with respect to the above 194 RB function signature was performed. Clustering was created based on the log ratio relative to the average for all 30 cell lines shown below.

The results are shown in FIG. In FIG. 1, the genes are arranged in the horizontal direction and the cell lines are arranged in the vertical direction. The expression level of each gene is represented by the log ratio (bottom 10) with respect to the average in all samples as in the above formula, and is shown in white when the expression level is higher than the average and black when it is lower.

Except for the cell line MDA-MB-468, it was revealed that normal and abnormal RB protein strains formed clusters (FIG. 1). That is, it was shown that RB function normal strain and abnormal strain can be separated by the 194 gene.

In order to evaluate the RB function prediction accuracy based on the RB function signature, leave-one-out cross validation was performed. That is, 1 sample data was removed from all samples, genes were selected from the remaining 29 sample data, and the RB function of 1 sample excluded by binary regression on the first singular value of singular value decomposition was predicted. As a result, the RB function state of 26 of 30 strains was correctly predicted, and the prediction accuracy was 87%.

<Development of RB status prediction method by gene expression of cyclin D1 and p16>
Next, in order to develop a method for predicting the presence or absence of RB dysfunction with a smaller number of genes, attention was paid to four genes involved in the p16-RB-E2F pathway in the RB function signature. The four genes are RB1 (RB gene), cyclin D1 (DCND1), p16, and GSK3B. The RB1 and cyclin D1 genes showed a positive correlation with the presence or absence of RB function (correlation coefficients were 0.61 and 0.66, respectively). On the other hand, both p16 and GSK3B are known to inhibit the CDK4 / cyclin D1 complex, but their gene expression was negatively correlated with RB function (correlation coefficient was -0.85, respectively). -0.61). Here, RB1, cyclin D1, p16, and GSK3B alone do not accurately identify the RB function.
However, RB function could be completely identified by taking the ratio of cyclin D1 and p16 gene expression (FIG. 2). At this time, the criteria for identification were as follows.
Normal RB function: Log ₁₀ (cyclin D1 / p16 gene expression ratio)> 0.6
RB dysfunction: Log ₁₀ (cyclin D1 / p16 gene expression ratio) <0.6
The cyclin D1 / p16 expression ratio was calculated as follows.

Also, the log ratio for HeLa S3 in a specific cell is calculated as follows.

By obtaining the cyclin D1 / p16 expression ratio defined as described above, it was revealed that a normal RB function strain and an abnormal RB function strain can be clearly distinguished (FIG. 2). That is, when the expression ratio of cyclin D1 / p16 is large (for example, when log ₁₀ (cyclin D1 / p16 gene expression ratio) is larger than 0.6), it can be determined that the RB function is normal, and the expression ratio of cyclin D1 / p16 is small. In the case (for example, when log ₁₀ (cyclin D1 / p16 gene expression ratio) is smaller than 0.6), it can be determined as an RB dysfunctional strain.

<PLK1 inhibitor induces RB status-dependent cell death in RB matched pair cell lines>
In order to confirm the RB status-dependent cell death inducing effect of the PLK1 inhibitor, it was examined whether the induction of apoptosis by the four types of PLK1 inhibitors occurs selectively only in RB dysfunction cell lines.

U-2 OS cell (U-2 OS RB shRNA strain) which caused dysfunction in RB by shRNA against RB, and U-2 OS cell (U-2 OS vector strain with normal RB function introduced with a vector as a control) ) Was administered a PLK1 inhibitor. The U-2 OS RB shRNA strain and the U-2 OS vector strain were prepared based on the description of Oncogene (2007) 26, 509-520. That is, the U-2 OS vector strain and the U-2 OS RB shRNA strain were cultured in the presence of a PLK1 inhibitor for 48 hours, and the proportion of cells that caused apoptosis after the culture was compared between the strains. As PLK1 inhibitors, four types of Compound Example 38 and Example 186 described in WO2006 / 049339 and Compound Example 5 and Example 81 described in WO2008 / 081914 were used. Apoptotic cells were identified by measuring the DNA content of the cells by flow cytometry. In this identification method, a cell having a DNA content less than that of a G1 phase cell (hereinafter referred to as subG1 cell) is defined as an apoptotic cell.
As shown in FIG. 3, all four PLK1 inhibitors were found to induce apoptosis selectively and strongly against RB shRNA strains with RB dysfunction compared to Vector strains with normal RB function. It was. From this, it was confirmed that the PLK1 inhibitor induces RB status-dependent cell death only in the RB dysfunctional strain.

<The gene expression ratio of cyclin D1 and p16 identifies RB status>
In order to confirm that the gene expression ratio of cyclin D1 and p16 can distinguish RB status, 10 cell lines with unknown RB status are prepared, and the cyclin D1 and p16 gene expression ratio of these cells and the actual RB status are calculated. Compared. The cell lines used are A427, NCI-H441, NCI-H23, NCI-H358, NCI-H661, NCI-H2030, NCI-H522, NCI-H1048, NCI-H2172, and NCI-H810. The gene expression levels of cyclin D1 and p16 in these cells were measured relative using a quantitative real-time PCR method.

Measurement of the expression levels of cyclin D1 and p16 by quantitative real-time PCR was performed as follows. First, the gene expression levels of cyclin D1 and p16 were measured by a real-time PCR method. That is, cells in the logarithmic growth phase were collected, and RNA was extracted and purified. Next, based on the obtained purified RNA, cDNA was synthesized using random primers, and the amount of cyclin D1 and p16 contained therein was determined with Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems). It was measured. The probe used for real-time PCR is TaqManaqGene Expression Assays (Applied Biosystems).

Here, relative quantification was performed using cDNA obtained from HeLaS3 cells in the same manner as a calibration sample. Therefore, the value of the obtained gene expression level is expressed as a relative ratio to the gene expression level in HeLaS3 cells and can be compared with the gene expression information used in Example 1. The cyclin D1 / p16 expression ratio was calculated in the same manner as in the method described in Example 1, and the RB status was calculated in the same manner as in Example 1 for cells with log ₁₀ (cyclin D1 / p16 expression ratio)> 0.6. RB function was predicted to be normal, and cells with log ₁₀ (cyclin D1 / p16 expression ratio) <0.6 were predicted to have RB function abnormality.

Further, the actual RB status was identified by the RB protein functional assay in the same manner as in Example 1. The actual RB status was identified by the RB protein functional assay as follows.

FIG. 5 shows a comparison between the predicted RB status, the value of log ₁₀ (cyclin D1 / p16 expression ratio), and the actual RB function. In FIG. 5, cells for which the RB status was correctly predicted by the gene expression ratio of cyclin D1 and p16 are indicated by ●, and cells for which the RB status was not predicted are indicated by ×.

As shown in FIG. 5, the RB function can be predicted with high accuracy from the cyclin D1 / p16 expression ratio. In particular, when log ₁₀ (cyclin D1 / p16 expression ratio) is larger than 0.6, the RB function is normal. It was confirmed that when the value is smaller than 0.6, the RB function can be predicted to be abnormal.
In addition, since the cell experiment system is a good model system that reflects clinical tissues, it is considered that the RB status identification method using the gene expression ratio of cyclin D1 and p16 can also be used for clinical samples. Therefore, it is possible to predict the RB function of cells in a clinical sample with high accuracy based on the cyclin D1 / p16 expression ratio.

<RB Status Predicted by Gene Expression Ratio of Cyclin D1 and p16 Correlates with Sensitivity to PLK1 Inhibitor>
To confirm that the sensitivity of the PLK1 inhibitor correlates with the RB status predicted from the gene expression ratio of cyclin D1 and p16, the PLK1 inhibitor (compound example 38 described in WO2006 / 049339 and described in WO2008 / 081914) The susceptibility test of lung cancer cells against Compound Example 5) was conducted, and the correlation between the obtained sensitivity information and the RB status predicted from the gene expression ratio of cyclin D1 and p16 was examined.

8 small cell carcinomas and 20 non-small cell carcinomas were used as lung cancer cells, and the RB status of these cells was predicted as shown in Table 6 from the gene expression ratio of cyclin D1 and p16. Here, the expression ratio of cyclin D1 and p16 was determined by the same method as in Example 1. Most small cell lung cancers were predicted to have RB dysfunction.

Sensitivity to the PLK1 inhibitor was determined by quantifying the number of cells after 72 hours of culturing in the presence of the PLK1 inhibitor using WST-8 (Kishida Chemical Co., Ltd.). Here, the complete growth inhibitory concentration (hereinafter referred to as IC ₁₀₀ ) was used as the sensitivity index. IC ₁₀₀ is the concentration of the compound that maintains the same number of cells after 72 hours of treatment with the compound as when the compound was added, and is an index reflecting the effect of cell death.

As shown in FIG. 6, it was confirmed that the sensitivity of the PLK1 inhibitor and the predicted RB status were correlated (Fischer's exact test, p value <0.05).

Claims (23)

1. Determining the expression level of the cyclin D1 gene and the expression level of the p16 gene in a patient-derived biological sample;
   determining the ratio of the expression level of the cyclin D1 gene to the expression level of the p16 gene;
   Predicting that the PLK1 inhibitor is effective when the ratio of the expression levels is smaller than that of the control cells.
2. The ratio of the expression levels is represented by the expression ratio of cyclin D1 / p16, and the value of the expression ratio of cyclin D1 / p16 is expressed by the following formula:
   

   

   The method of claim 1 represented by:
3. Determining the expression level of the cyclin D1 gene and the expression level of the p16 gene in a patient-derived biological sample;
   determining the ratio of the expression level of the cyclin D1 gene to the expression level of the p16 gene;
   Predicting whether the RB function in the cell is abnormal, including predicting that the RB function in the cell in the biological sample is abnormal when the ratio of the expression levels is small compared to the control cell how to.
4. The ratio of the expression levels is represented by the expression ratio of cyclin D1 / p16, and the value of the expression ratio of cyclin D1 / p16 is expressed by the following formula:
   

   

   The method of claim 3 represented by:
5. Predicting whether or not RB function in cells in the biological sample is abnormal based on RB gene mutation, RB gene expression level and / or RB protein production level in a biological sample derived from a patient;
   Predicting the effectiveness of the PLK1 inhibitor when the RB function is abnormal, and predicting that the PLK1 inhibitor is effective.
6. Determining the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a patient-derived biological sample;
   When the expression level of the gene described in FIG. 7 is smaller than that of the control cell and / or when the expression level of the gene described in FIG. 8 is higher than that of the control cell, the PLK1 inhibitor is effective. Predicting the effect of a PLK1 inhibitor.
7. The expression level of one or more genes described in FIGS. 7 and 8 is determined, and the gene described in FIG. 7 includes the cyclin D1 gene, and the gene described in FIG. 8 includes the p16 gene. the method of.
8. Determining the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a patient-derived biological sample;
   When the expression level of the gene described in FIG. 7 is smaller than that of the control cell and / or when the expression level of the gene described in FIG. 8 is higher than that of the control cell, the RB function in the cell is abnormal. Predicting whether or not the RB function in the cell is abnormal.
9. The expression level of one or more genes described in FIGS. 7 and 8 is determined, and the gene described in FIG. 7 includes the cyclin D1 gene, and the gene described in FIG. 8 includes the p16 gene. the method of.
10. 10. The method according to any one of claims 1 to 9, wherein the patient-derived biological sample contains cancer cells.
11. 10. The method according to any one of claims 1 to 9, wherein the patient-derived biological sample contains lung cancer cells.
12. The method according to any one of claims 1 to 9, wherein the patient-derived biological sample contains small cell carcinoma cells.
13. A pharmaceutical composition for treating a cancer patient having cancer cells with abnormal RB function, the pharmaceutical composition comprising a pharmaceutically effective amount of a PLK1 inhibitor.
14. A drug for inducing cell death in cells with abnormal RB function, comprising a pharmacologically effective amount of a PLK1 inhibitor.
15. The pharmaceutical composition according to claim 13, wherein the cancer cell is predicted to have an abnormal RB function by the method according to any one of claims 3, 4, 8 or 9.
16. The drug according to claim 14, wherein the cell is predicted to have abnormal RB function by the method according to any one of claims 3, 4, 8, or 9.
17. Determining the expression level of the cyclin D1 gene and the expression level of the p16 gene in a patient-derived biological sample;
    determining the ratio of the expression level of the cyclin D1 gene to the expression level of the p16 gene;
    Administering a PLK1 inhibitor to a patient when the ratio of the expression levels is smaller than that of control cells.
18. The ratio of the expression levels is represented by the expression ratio of cyclin D1 / p16, and the value of the expression ratio of cyclin D1 / p16 is expressed by the following formula:
    

    

    The method of claim 17 represented by:
19. Predicting whether or not RB function in cells in the biological sample is abnormal based on RB gene mutation, RB gene expression level and / or RB protein production level in a biological sample derived from a patient;
    Administering a PLK1 inhibitor to a patient when the RB function is abnormal.
20. Determining the expression level of one or more genes described in FIG. 7 and / or the expression level of one or more genes described in FIG. 8 in a patient-derived biological sample;
    When the expression level of the gene described in FIG. 7 is small compared to the control cells and / or when the expression level of the gene described in FIG. 8 is large compared to the control cells, a PLK1 inhibitor is administered to the patient. A method of treating cancer, comprising: administering.
21. 21. The expression level of one or more genes described in FIGS. 7 and 8 is determined, the gene described in FIG. 7 includes a cyclin D1 gene, and the gene described in FIG. 8 includes a p16 gene. the method of.
22. One or more isolated nucleic acid sequences selected from the group consisting of FIG. 7 and FIG. 8 that are associated with RB function.
23. 23. An array comprising nucleic acids that bind to one or more isolated nucleic acid sequences of claim 22.

Images