KR102346174B1 - Method for providing the information for selecting the drugs for treating EML4-ALK positive non-small cell lung cancer and compositions for treating non-small cell lung cancer resistant to ALK inhibitors - Google Patents

Abstract

The present invention relates to a method for providing information for selecting a therapeutic agent for EML4-ALK-positive non-small cell lung cancer patients and a composition for treating ALK inhibitor-resistant non-small cell lung cancer. A method for providing information for selecting a treatment drug for EML4-ALK-positive non-small cell lung cancer patients, comprising measuring the expression level and methylation level, and a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient It relates to a composition for treating non-small cell lung cancer that has acquired resistance to an ALK inhibitor.

Description

TECHNICAL FIELD The present invention relates to a method for providing information for selecting a treatment drug for a patient with EML4-ALK positive non-small cell lung cancer, and a composition for treating ALK inhibitor-resistant non-small cell lung cancer. compositions for treating non-small cell lung cancer resistant to ALK inhibitors}

The present invention relates to a method for providing information for selecting a therapeutic agent for EML4-ALK-positive non-small cell lung cancer patients and a composition for treating ALK inhibitor-resistant non-small cell lung cancer. A method for providing information for selecting a treatment drug for EML4-ALK-positive non-small cell lung cancer patients, comprising measuring the expression level and methylation level, and a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient It relates to a composition for treating non-small cell lung cancer that has acquired resistance to an ALK inhibitor.

ALK (Anaplastic Lymphoma kinase) is a tyrosine kinase receptor, Ras/Raf/MEK/ERK1/2 pathway, JAK (Janus kinase)/STAT (signal transducers and activators of transcription) pathway, Pl3K (Phosphatidylinositol) It plays an important role in cell proliferation, growth and survival through the 3-kinase)/Akt pathway. However, overexpression of ALK leads to abnormal proliferation and growth of cancer cells.

On the other hand, in non-small cell lung cancer, EML4 (echinoderm microtubule-associated protein-like 4)-ALK mutation of ALK is known to play an important role in abnormal proliferation of cancer cells. Research and interest in cancer treatment is growing.

ALK inhibitors such as crizotinib and ceritinib have been used as targeted therapeutics for ALK. There is an invisible problem. In fact, only about 10% of patients with non-small cell lung cancer are responsive to these drugs.

Therefore, there is a need to develop a new therapeutic agent for treating non-small cell lung cancer that has acquired resistance to an ALK inhibitor as well as an efficient method for accurately determining whether to use an ALK inhibitor in a patient group of EML4-ALK-positive non-small cell lung cancer.

Accordingly, the present inventors conducted intensive research to develop a new therapeutic agent for treating non-small cell lung cancer that has acquired resistance to an ALK inhibitor in a patient group of EML4-ALK-positive non-small cell lung cancer. deaminase) was found to be decreased and the expression level was high, and deoxycytidine was epigenetically oxidized to deoxycytidine in CDA overexpressed ALK inhibitor-resistant non-small cell lung cancer. It was found that the analogue exhibits an excellent cancer cell growth inhibitory effect, and the present invention was completed.

Accordingly, it is an object of the present invention to provide information for selecting a therapeutic drug for EML4-ALK-positive non-small cell lung cancer patients, including an agent for measuring the expression level of CDA (cytidine deaminase) protein or gene, or the methylation level of CDA gene is to provide

Another object of the present invention is to provide information for drug selection for EML4-ALK-positive lung cancer patients, the expression level of CDA (cytidine deaminase) protein or gene from a biological sample provided from EML4-ALK-positive lung cancer patients, or CDA It is to provide an information providing method for selecting a drug for treatment of EML4-ALK-positive non-small cell lung cancer patients, including measuring the methylation level of the gene.

Another object of the present invention is a pharmaceutical composition for preventing or treating EML4-ALK-positive non-small cell lung cancer that has acquired resistance to an ALK inhibitor, comprising a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient is to provide

In order to achieve the above object of the present invention, the present invention provides a therapeutic drug for EML4-ALK-positive non-small cell lung cancer patients comprising an agent for measuring the expression level of a CDA (cytidine deaminase) protein or gene, or the methylation level of a CDA gene Compositions for providing information for selection are provided.

In order to achieve another object of the present invention, in order to provide information for drug selection for EML4-ALK-positive lung cancer patients, the present invention provides CDA (cytidine deaminase) protein or It provides an information providing method for selecting a drug for treatment of EML4-ALK-positive non-small cell lung cancer patients, comprising measuring the expression level of the gene, or the methylation level of the CDA gene.

In order to achieve another object of the present invention, the present invention provides EML4-ALK-positive non-small cell lung cancer that has acquired resistance to an ALK inhibitor comprising a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for prevention or treatment is provided.

Hereinafter, the present invention will be described in detail.

According to an embodiment of the present invention, in the EML4-ALK-positive non-small cell lung cancer cell line that is resistant to the ALK inhibitor, the methylation level in the CDA (cytidine deaminase) gene is reduced compared to the cell line that does not show resistance to the ALK inhibitor, and as a result, It was confirmed that the expression level of the CDA gene was significantly increased. Therefore, by detecting the expression level of the CDA gene or protein, or the methylation level of the CDA gene in a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient, it can be determined whether resistance to an ALK inhibitor will be exhibited.

Accordingly, the present invention provides a composition for providing information for selecting a treatment drug for patients with EML4-ALK-positive lung cancer, comprising an agent for measuring the expression level of a CDA protein or gene, or the methylation level of a CDA gene.

The CDA refers to an enzyme having an activity to convert cytidine (or deoxycytidine) to uridine (or deoxyuridine), and CDA (cytidine deaminase; EC number 3.5.4.5; eg, CDD): GenBank Accession Nos. Sequences such as NP_001776.1 (gene: NM_001785.2), CAA06460.1 (gene: AJ005261.1), NP_416648.1 (gene: NC_000913.3) may be referred to.

Preferably, in the present invention, the CDA protein may include the amino acid sequence of SEQ ID NO: 1 and the CDA gene may include the nucleotide sequence of SEQ ID NO: 2.

[SEQ ID NO: 1]

maqkrpactl kpecvqqllv csqeakksay cpyshfpvga alltqegrif kgcnienacy

plgicaerta iqkavsegyk dfraiaiasd mqddfispcg acrqvmrefg tnwpvymtkp

dgtyivmtvq ellpssfgpe dlqktq

[SEQ ID NO: 2]

a tggcccagaa gcgtcctgcc tgcaccctga agcctgagtg tgtccagcag ctgctggttt

gctcccagga ggccaagaag tcagcctact gcccctacag tcactttcct gtgggggctg

ccctgctcac ccaggagggg agaatcttca aagggtgcaa catagaaaat gcctgctacc

cgctgggcat ctgtgctgaa cggaccgcta tccagaaggc cgtctcagaa gggtacaagg

atttcagggc aattgctatc gccagtgaca tgcaagatga ttttatctct ccatgtgggg

cctgcaggca agtcatgaga gagtttggca ccaactggcc cgtgtacatg accaagccgg

atggtacgta tattgtcatg acggtccagg agctgctgcc ctcctccttt gggcctgagg

acctgcagaa gacccagtga

In the present invention, the "EML4-ALK-positive non-small cell lung cancer patient" refers to a patient with ALK gene mutation among non-small cell lung cancer patients. The mutation of the ALK gene may be formed by fusion of EML4 and ALK gene. The fused gene induces cancer by expressing EML4-ALK.

After determining whether resistance to an ALK inhibitor has been acquired by measuring the expression level of the CDA protein or gene or the methylation level of the CDA gene in a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient through the composition of the present invention, treatment It can provide information for drug selection.

Specifically, as a result of analyzing the expression level of the CDA protein or gene or the methylation level of the CDA gene in a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient through the composition of the present invention, resistance to the ALK inhibitor was not acquired. If the expression level of the CDA protein or gene is increased or the methylation level of the CDA gene is decreased compared to the patient group without the ALK inhibitor, the patient can be judged as a patient who has acquired resistance to the ALK inhibitor. Selection as a therapeutic drug can be excluded.

Conversely, as a result of analyzing the expression level of the CDA protein or gene or the methylation level of the CDA gene in a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient through the composition of the present invention, the patient group that did not acquire resistance to the ALK inhibitor If the expression level of the CDA protein or gene is decreased or there is no change compared to Therefore, ALK inhibitors can be selected as therapeutic agents.

Alternatively, after administration of an ALK inhibitor to an EML4-ALK-positive non-small cell lung cancer patient for a certain period of time, the expression level of a CDA protein or gene in a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient through the composition of the present invention, or CDA By analyzing the methylation level of the gene, it is possible to make a judgment on whether to continue to administer the ALK inhibitor.

In the present invention, the "ALK inhibitor" is a drug that has a therapeutic effect on the EML4-ALK-positive non-small cell lung cancer patient, and refers to a drug that inhibits the kinase activity of EML4-ALK. In the present invention, whether or not resistance to an ALK inhibitor is acquired is determined when an ALK inhibitor is administered for a certain period of time for the treatment of non-small cell lung cancer to a non-small cell lung cancer patient, compared with the initial drug response to which the drug is administered. ALK inhibitors when the cancer symptoms show no improvement, alleviation, alleviation or treatment symptoms due to extremely low sensitivity to ALK inhibitors after It can be said that independent tolerance has been acquired. In particular, when resistance to an ALK inhibitor is acquired as described above, an increase in the expression level of a CDA gene or protein or a decrease in CDA gene methylation can be said to be an important factor in inducing resistance. The progression of non-small cell lung cancer can be confirmed by any means that can be used to image the development of cancer, for example, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, X-ray imaging, mammography, PET scan, radioactive As long as it can be confirmed through imaging techniques such as a nuclide scan and a bone scan, the means is not particularly limited.

In the present invention, the ALK inhibitor may be selected from the group consisting of crizotinib, ceritinib, alectinib, brigatinib and entrectinib, but in this It is not limited.

In the present invention, the term 'expression' means that a protein or nucleic acid is produced in a cell.

In the present invention, the term 'protein' is used interchangeably with 'polypeptide' or 'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in a natural state.

As used herein, the term 'polynucleotide' or 'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included. 'mRNA' is an RNA that delivers genetic information (gene-specific nucleotide sequence) to the ribosome, which specifies the amino acid sequence from a specific gene during protein synthesis.

In the present invention, the agent is an antibody or aptamer that specifically binds to a CDA protein; Alternatively, it may be a primer or probe that specifically binds to the CDA gene.

As used herein, the term 'antibody' refers to an immunoglobulin that specifically binds to an antigenic site. The CDA antibody in the present invention is an antibody that specifically binds only to a CDA protein without reacting to any other protein other than CDA. In the present invention, the antibody that specifically binds to the CDA protein may preferably be an antibody that specifically binds to the protein (CDA) comprising the amino acid sequence represented by SEQ ID NO: 1. The antibody may be prepared according to a conventional method in the art, such as cloning a CDA-encoding gene into an expression vector to obtain a protein encoded by the gene, and injecting the obtained protein into an animal to obtain an antibody produced. can The CDA antibody may be prepared using the full-length CDA sequence protein, or may be prepared using a polypeptide fragment including the antigenic region of the CDA protein. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody or a monoclonal antibody. In addition, a part of the whole antibody is also included in the antibody of the present invention as long as it has antigen-antibody binding (reaction), and all kinds of immunoglobulin antibodies that specifically bind to CDA are included. For example, a functional fragment of an antibody molecule as well as a complete antibody having two full-length light chains and two full-length heavy chains, i.e. Fab, F(ab'), F(ab') with antigen-binding function. 2 and Fv, and the like. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to CDA protein.

In the present invention, the term 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample. , it is possible to specifically confirm the presence of the target protein in the sample. According to a general method for preparing an aptamer, the aptamer is synthesized by determining the sequence of an oligonucleotide having a selective and high binding affinity to the target protein (CDA protein in the present invention) to be confirmed, and then 5' of the oligonucleotide. It may be accomplished by modifying the terminal or the 3' terminal with -SH, -COOH, -OH or NH2 so as to bind to the functional group of the aptamer chip, but is not limited thereto.

In the present invention, measuring the expression level of the CDA gene includes measuring the expression level of transcripts derived from the CDA gene, and for example, measuring the CDA mRNA expression level.

Therefore, the agent for measuring the expression level of the CDA gene is not limited thereto, but may preferably refer to an agent for detecting mRNA expressed from the gene. Therefore, the type of the agent for detecting the CDA gene is not particularly limited as long as it is a ligand that specifically attaches or hybridizes to the mRNA expressed from the gene, but may be, for example, a primer (pair) or a probe.

The "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and refers to a short nucleic acid sequence that serves as a starting point for template strand copying. . The primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.

The sequence of the primer does not need to have a completely complementary sequence to a partial nucleotide sequence of the template, and it is sufficient if it hybridizes with the template and has sufficient complementarity within a range capable of performing an intrinsic function of the primer. Therefore, in the present invention, the primer for measuring the expression level of CDA mRNA does not need to have a perfectly complementary sequence to the CDA gene sequence, and the amount of CDA mRNA is determined by amplifying a specific section of CDA mRNA or CDA cDNA through DNA synthesis. It is sufficient if it has a length and complementarity suitable for the purpose of measurement. The primers for the amplification reaction are composed of a set (pair) that complementarily binds to the template (or sense) and the opposite side (antisense) of both ends of a specific section of the CDA mRNA to be amplified. Primers can be easily designed by those skilled in the art by referring to the CDA mRNA or cDNA sequence.

A 'probe' refers to a fragment of a polynucleotide such as RNA or DNA with a length of several to several hundred base pairs that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. In addition, since it is labeled, the presence or absence of the target mRNA or cDNA to bind to, the amount of expression, etc. can be confirmed. For the purpose of the present invention, a probe complementary to CDA mRNA may be used for diagnosis of vascular dementia by performing a hybridization reaction with a sample of a subject and measuring the expression level of CDA mRNA. Probe selection and hybridization conditions can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within the range that does not interfere with hybridization with CDA mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.

In order to measure the expression level of the CDA protein of the composition of the present invention, an antibody, an aptamer, or a primer, a probe that selectively recognizes a CDA protein as a marker, or a primer, a probe that recognizes a CDA mRNA as a marker, as well as one or more other suitable analysis methods A component composition, solution or device may be included.

In the present invention, the 'methylation' results in gene inactivation without altering the DNA sequence, and is classified as an epigenetic change in distinction from genetic changes such as point mutations and gene deletions. In the present invention, the change in the methylation level includes both hypermethylation and hypomethylation.

In the methylation, a methyl group (-CH3) is bonded to the 5' carbon region of CpG by DNA methyl transferase (DNMT). CpG islands with several CpG dinucleotides are mainly located near promoters or exon start sites. When a methyl group binds to CpG islands, mRNA transcription is disturbed and gene expression is suppressed.

Accordingly, in the present invention, the CDA gene methylation level may be the methylation level at the promoter or enhancer region of the CDA gene.

In the present invention, the 'agent for measuring the methylation level' may include a primer specific for the methylated allele sequence of the CDA gene and a primer specific for the unmethylated allele sequence. In addition, the preparation of the present invention may include a primer pair and an extension primer specific for the allele sequence of the CDA gene.

The primer may be preferably designed according to the sequence of the CpG island to be analyzed for methylation, and a pair of primers capable of specifically amplifying cytosine that is methylated and not modified by bisulfite and is not methylated It may be a primer pair capable of specifically amplifying a cytosine modified by bisulfite. It may also be a primer pair and an extension primer specific for the modified sequence.

In addition, the agent for measuring the methylation level of a gene in the present invention may include a compound that modifies an unmethylated cytosine base, a primer pair specific for the modified sequence of the gene, and an extension primer.

The compound that modifies the unmethylated cytosine base may be, but is not limited to, bisulfite, preferably sodium bisulfite. A method for detecting methylation of a promoter by modifying an unmethylated cytosine residue using such a bisulfite is well known in the art (Herman JG et al., 1996, Proc. Natl. Acad. Sci. USA). , 93: 9821 - 9826; WO01/26536; US2003/0148326A1).

In addition, the methylation-sensitive restriction enzyme is a restriction enzyme capable of specifically detecting methylation of CpG islands, and is preferably a restriction enzyme containing CG as a recognition site for the restriction enzyme. Examples include, but are not limited to, SmaI, SacII, EagI, HpaII, MspI, BssHII, BstUI, NotI, and the like. Depending on the methylation or unmethylation at C of the restriction enzyme recognition site, whether or not cleavage by the restriction enzyme is changed, which can be detected through PCR or Southern blot analysis. Methylation-sensitive restriction enzymes other than the above restriction enzymes are well known in the art.

According to one embodiment of the present invention, the composition may be provided in the form of a kit for providing information for selecting a treatment drug for a patient with EML4-ALK positive non-small cell lung cancer.

The kit may further comprise one or more other component compositions, solutions or devices suitable for the assay method. In a specific embodiment, the kit may be a kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer specific for a gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another aspect, it may preferably be a kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescently labeled probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

The present invention also provides information for selecting a drug for treatment of EML4-ALK-positive non-small cell lung cancer patients, the expression level of a CDA (cytidine deaminase) protein or gene from a biological sample provided from an EML4-ALK-positive non-small cell lung cancer patient; Or, it provides an information providing method for selecting a drug for treatment of EML4-ALK-positive non-small cell lung cancer patients, comprising the step of measuring the methylation level of the CDA gene.

In the present invention, the term 'measurement' in the present invention may preferably mean 'analysis', and the analysis may be qualitative or quantitative analysis. The qualitative analysis may mean measuring and confirming the presence of a target substance, and the quantitative analysis may mean measuring and confirming a change in the presence level (expression level) or amount of the target substance. have. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and preferably quantitative measurement may be performed.

In the present invention, the sample may be used without limitation as long as it is obtained from a patient with EML4-ALK-positive non-small cell lung cancer. For example, cancer cells or tissues, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, and various secretions obtained by biopsy, etc. , urine, feces, etc. Preferably, a biological liquid sample (body fluid sample) may be used, for example, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical or vaginal secretion, urine and cerebrospinal fluid. it could be

The sample may be pretreated prior to use for analysis or measurement. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.

In the present invention, the CDA protein expression level measurement is not particularly limited as long as it is performed by a protein expression measurement method known in the art, for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Oukteroni It may be carried out by a method selected from the group consisting of immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation, complement fixation, FACS, and protein chip.

In the present invention, the measurement of the gene expression level is not particularly limited as long as the measurement method is by a gene expression measurement method known in the art, for example, PCR, RNase protection assay, Northern blotting, Southern blotting ( southern blotting) and a method selected from the group consisting of DNA chips.

In the present invention, the measurement of the methylation level of the gene is not particularly limited as long as it is by a gene methylation measurement method known in the art, for example, PCR, methylation specific PCR, real-time methylation specific PCR (real). time methylation specific PCR), PCR using a methylated DNA-specific binding protein, quantitative PCR, DNA chip, pyrosequencing, and bisulfite sequencing may be performed by a method selected from the group consisting of.

As a result of measuring the expression level of the CDA protein or gene or the methylation level of the CDA gene in a biological sample provided from a specific patient according to the method of the present invention, the expression level of the CDA (cytidine deaminase) protein or gene is higher than that of the control group Or, when the methylation level of the CDA gene is lower than that of the control group, it can be determined that the non-small cell lung cancer patient's responsiveness to the ALK inhibitor is low. That is, since the patient may be a non-small cell lung cancer patient who is resistant to an ALK inhibitor, it may be desirable to select a therapeutic agent other than the ALK inhibitor to establish a treatment strategy.

The present invention also provides a pharmaceutical composition for preventing or treating EML4-ALK-positive non-small cell lung cancer that has acquired resistance to an ALK inhibitor, comprising a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient do.

According to an embodiment of the present invention, as a result of treatment with a deoxycytidine analog in a non-small cell lung cancer cell line that is resistant to an ALK inhibitor, it was confirmed that apoptosis of cancer cells was induced.

In the present invention, the type of the deoxycytidine analog is not particularly limited, and for example, 5hmdC (5-hydroxymethyl-2'-deoxycitidine), 5fdC (5-formyl-2'-deoxycytidine), 5cadC (5- carboxyl-2'-deoxycitidine) and the like (Nature. 2015;524(7563):114-8).

Preferably, in the present invention, the deoxycytidine analog may be 5hmdC or 5fdC.

According to an embodiment of the present invention, EML4-ALK-positive non-small cell lung cancer, which has acquired resistance to an ALK inhibitor, overexpresses CDA, and CDA converts 5hmdC and 5fdC into uridine variants. This converted uridine mutant is inserted into DNA, accumulates DNA damage, and can eventually induce apoptosis.

Accordingly, in the present invention, the pharmaceutical composition can be particularly effective in non-small cell lung cancer in which CDA protein or mRNA expression is increased, or CDA gene methylation level is decreased.

The deoxycytidine analog according to the present invention may be used as such or in the form of a pharmaceutically acceptable salt. As used herein, 'pharmaceutically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not normally cause allergic reactions or similar reactions when given to humans. Acid addition salts formed with free acids are preferred. The free acid may be an organic acid or an inorganic acid. The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

The pharmaceutical composition according to the present invention may contain a deoxycytidine analog or a pharmaceutically acceptable salt thereof alone or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and additionally contain an excipient or diluent. can do. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, various drug delivery materials used for oral administration may be included. In addition, the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. For other pharmaceutically acceptable carriers and agents, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention may be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. can be

The pharmaceutical composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the administration route as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. using methods known in the art. can For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, grinding it, adding suitable adjuvants, and processing it into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches, including corn starch, wheat starch, rice starch and potato starch, cellulose, Cellulose, including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, and the like, fillers such as gelatin, polyvinylpyrrolidone, and the like may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant if necessary. Furthermore, the pharmaceutical composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, and a preservative.

Formulations for parenteral administration may be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, which is a commonly known recipe for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per kg of the patient's body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route and number of treatments, as well as the patient's age, weight, health status, sex, severity of disease, diet and excretion rate, etc., the effective dosage for the patient is determined. Therefore, in consideration of this point, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.

In one embodiment of the present invention, the composition may be administered in combination with an ALK inhibitor.

When the composition of the present invention is administered in combination with an ALK inhibitor, the composition may be administered separately in the form of a single composition or may be administered simultaneously in the form of a combination drug. That is, the composition may be administered simultaneously (simultaneous), separately (separate) or sequentially (sequential) with the ALK inhibitor, and there is no particular limitation on the order of administration when sequentially administered.

According to the composition and method provided by the present invention, it is possible to accurately distinguish EML4-ALK-positive non-small cell lung cancer patients who are resistant to ALK inhibitors. can

In addition, the composition of the present invention comprising a deoxycytidine analog may exhibit excellent anticancer effects in patients with EML4-ALK-positive non-small cell lung cancer who are resistant to ALK inhibitors.

1 including FIGS. 1A to 1F is a result confirming the change in DNA methylation level in H3122 and LR cells. (A) Experimental workflow for characterizing resistance-acquired cells. LR cells, which are ceritinib-resistant cells, were established by treatment with increasing concentrations of ceritinib. (B) Heatmap showing differentially expressed genes (DEG) compared to LR and H3122. (C) Analysis of relative mRNA expression of DNA methyltransferase (DNMT) and Ten-eleven translocation (TET) families by qRT-PCR. (D) Heatmap of differentially methylated loci between H3122 and LR cells at 41,223 CpG. (E) Correlation of differences in DNA methylation and mRNA expression levels between H3122 and LR. (F) Gene set analysis for differentially methylated genes in the KEGG pathway and biological processes.
Figure 2 is the result of confirming the change of DNA methylation and mRNA expression in naive (naive) and resistant cell lines. DNA methylation levels of CDA were plotted as pie charts representing methylation rates (black) in individual Infinium Human Methylation 450K BeadChip probes (red lines). cg04087271, cg20619374 and cg06984156 for CDA. Graphs are relative fold changes in mRNA levels of CDA in H3122 and LR cell lines.
FIG. 3 including FIGS. 3A to 3F shows the results of single cell RNA sequence analysis from H3122 and LR cells. (A) t-SNE plots showing scRNA-seq results of H3122 (blue, n = 4,977) and LR (red, n = 2,250) cells. (B) t-SNE visualizing 10 clusters (C0 to C9) from H3122 and LR cells. (C) Proportion of H3122 and LR cells in clusters. (D) Proportion of cell cycle status of H3122 and LR cells in cluster 10. (E) Trajectory analysis of H3122 and LR cells shown in two dimensions (top) and highlighted in ten clusters. (F) Heatmap of differentially expressed genes in whole cells between H3122 and LR.

FIG. 4 including FIGS. 4A to 4C is a result showing single cell expression analysis of CDA in H3122 and LR cell populations (A) CDA expression in t-SNE map. (B) Violin plots showing the expression of CDA in each cluster. Each dot represents gene expression in each single cell. (C) Expression heatmap showing expression of CDA between naive and resistant states over time (cells are sorted by time and colors indicate expression levels).
5 and 6 including FIGS. 5A to 5C are results of evaluating the cytotoxicity of 5hmdC in CDA overexpressing cells (A) CDA mRNA expression level (top) and protein expression level in H3122, LR, H1299 and H1703 cells (lower). (B) Colony formation of H3122, LR, H1299 and H1703 cells treated with 5hmdC for 10 days. (C) Cell growth curves of H3122, LR, H1299 and H1703 cells treated with 5hmdC (0 μM, black; 1 μM, blue; 5 μM, magenta; 10 μM, red) for the indicated times. (Figure 6) Immunofluorescence of γH2AX in H3122, LR, H1299 and H1703 cells (green). DMSO and 5hmdC (10 uM) were treated. Nuclear DNA was stained with DAPI (blue).
7 including FIGS. 7A to 7C is a result confirming that CDA expression is related to overall survival of lung adenocarcinoma (LUAD) patients based on the TCGA database. (A) Expression levels of CDA in LUAD samples (n = 483) and normal tissue samples (n = 347). (B) Analysis of overall survival rates in patients with low or high CDA expression levels by the Kaplan-Meier method and the log-rank test in LUAD. (C) Model for targeting CDA with 5hmdC to overcome acquired resistance to ceritinib. During acquired resistance to ceritinib, CDA methylation is reduced and CDA expression is increased. Epigenetic nucleosides such as 5hmdC can induce DNA damage and apoptosis in CDA overexpressing resistant cells.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Experimental method

1. cell line

Lung cancer cell lines, H3122, H1299 and H1703, were treated with 1% antibiotics (Gibco, Cat # 15240062 Thermo Fisher Scientific, Waltham, MA) and 10% fetal bovine serum (HyClone, UT, USA). Cultured in RPMI-1640 medium (WELGENE, Cat number LM 011-01, Korea) supplemented with Ceritinib (LDK378). Resistant LR cells have been established in previous studies. Briefly, H3122 cells were _{cultured by gradually increasing the concentration starting with IC 30} of ceritinib, and resistant cells were induced after approximately 6 months of culture under continuous 1 μM ceritinib treatment conditions. All cells were maintained at 37° C. in a humidified atmosphere containing 5% CO _{2 .}

2. RNA sequencing analysis (Bulk RNA-seq analysis)

RNA-Seq libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA), and sequencing was performed using the Illumina HiSeq2000 platform to generate 100 bp paired-end reads. The sequenced reads were mapped to the human genome (hg19) using STAR (v.2.5.1), and gene expression levels were quantified with a count module in STAR. The edgeR (v.3.12.1) package was used to select differentially expressed genes (DEGs) from RNA-seq count data. Meanwhile, the trimmed mean of M-values normalization (TMM) normalized the CPM (number per million) of each gene and log2-transformed for further analysis.

3. Gene Set and Pathway Analysis

Gene set and pathway analysis was performed for DEGs associated with DNA methylation changes using DAVID (v.6.8). Significantly enriched pathways were obtained using a cutoff threshold of p-value <0.05. Only gene sets rich in at least three mutated genes were considered for further analysis.

4. qPCR analysis

RNA was isolated using the RNeasy Plus Mini Kit (Qiagen #74136) according to the manufacturer's instructions. cDNA was synthesized from 1 μg RNA using the iScript cDNA Synthesis Kit (Bio-Rad, Cat. No. 1708890). PCR reactions were performed in triplicate for each sample using 200 ng of cDNA, 7.5 μl of SYBR GREEN, and 0.3 μl of each primer. Gene expression values were normalized to beta-actin.

5. DNA Methylation Analysis

DNA methylation analysis of H3122 and LR cells was analyzed in duplicate using an Illumina Infinium methylated 450K bead chip array (San Diego, CA, USA) according to the manufacturer's protocol. The resulting DNA methyl/non-methyl signal intensity data were brought into R (v.3.4.2) for analysis. Normalization was performed using the SWAN method with background correction using minfi R package 3, v.1.30.0. Probes with detection p-values <0.05 and DNA methylation values were summarized as normalized mean beta values. For identification and extraction of differentially methylated regions (DMR) between H3122 and LR, statistical analysis was performed using DMRcate 4, v.1.20.0 R package. DMR was selected for probes with p values <0.05 and mean differences greater than 15%.

6. Single-cell RNA-seq library preparation and sequencing

Samples were prepared as outlined in the 10x Genomics Single Cell 3' v2 Reagent Kit Instructions for Use. Chromium Single Cell 3' Library and Gel Bead Kit V2 (PN-120237), Chromium Single Cell 3' Chip Kit V2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used with 10x Genomics Chromium. Samples were sequenced on a HiSeq 2500 using the following run parameters. Read 1-26 cycles, read 2-98 cycles, index 1-8 cycles. A median sequencing depth of 60,000 reads/cell was targeted for each sample.

7. Single-cell RNA-seq data processing and analysis

The pipeline served by the 10X genome was followed after pretreatment. The process of converting raw sequencing data (bcl files) into fastq and fitting to the human reference genome hg19 was performed using Cell Ranger software. A raw gene expression matrix was generated using the Cell Ranger Count module. To aggregate H3122 and LR data, Cell Ranger's 'aggr' module was used without the 'normalize' option. The rest of the process was performed using the guided analysis pipeline in R (https://www.R-project.org/) and Seurat 5, v.2.4.

Cell and gene filtration processes included only genes expressed in at least 3 cells and cells expressed in at least 200 genes. Cells with a mitochondrial genome transcript ratio greater than 50% were considered lysed and excluded. For data normalization, the global-scaling normalization method 'LogNormalize' was used with default parameters. Selection of variable genes, data scaling, and linear dimension reduction steps were performed sequentially according to the Seurat-guided analysis pipeline. To determine the 'dimension' of the data set, the elbow plot was checked and 10 pcs were determined. Cells were clustered using Seurat's 'FindClusters' module and clusters were visualized using t-Distributed Stochastic Neighbor Embedding (tSNE) implemented in Seurat. I used the 'FindAllMarkers' module to find DEGs in each cluster.

8. Western Blot

Cells were lysed in Laemmli Lysis-buffer (4% (w/v) SDS, 20% glycerol, 120 mM Tris-Cl (pH 6.8)) and supplemented with a protease inhibitor (Roche, Cat. No. 5892791001). Protein concentrations were determined using the Bradford Protein Assay Kit (BioRad). The protein was boiled at 100 °C for 5 min. Proteins were loaded onto 10-15% SDS-PAGE gels and transferred to PVDF membranes (Roche, Cat. No. 3010040001). After blocking with 5% skim milk (BD, Cat. No. 232100) or 5% BSA (Sigma-Aldrich, Cat. No. A7906) with TBS-T, membranes were incubated with the indicated primary antibodies overnight at 4 °C. . After washing with TBS-T, membranes were incubated for 30 min at room temperature for secondary antibody reaction (goat anti-mouse, Invitrogen, 31430; goat anti-rabbit, Invitrogen, 31460; 1:5000 ratio). After washing with TBS-T, blots were detected by ECL kit (Advansta, Cat. No. K-12045-D50) and visualized using a Fujifilm LAS-4000.

9. Colony Formation Assay

Harvested cells were counted in a Countess automated cell counter (Invitrogen, Carlsbad, CA) and seeded in 6 well plates (Corning, Cat. No. 3506). Plates were incubated at 37°C in 5% CO2. Cells were fixed and stained with 0.5% crystal violet, 3.7% formaldehyde and 30% ethanol. All analyzes were performed in triplicate and repeated in at least two independent experiments.

10. Cell Differentiation Assay

Harvested cells were counted in a Countess automated cell counter and plated in 24-well plates (Corning, Cat. No. 3524). Micrographs were taken every 2 h using an IncuCyte ZOOM system (Essen Biosciences), and confluence of cultures was measured using Incucyte software (Essen Biosciences) over 160 h.

11. Immunofluorescence staining for gammaH2A.X

Cells were plated in 8 wells (Lab-Tek II, Cat. No. 15446) and incubated at 37° C. in 5% CO 2 . Cells were washed twice with cold DPBS (WELGENE, Cat. No. LB001-02) and fixed with 4% paraformaldehyde (Biosesang, Cat. No. P2031) for 2 h at room temperature. After three washes in cold DPBS, cells were permeabilized in 0.25% Triton X-100 (Sigma Aldrich, Cat. No. T9284) for 10 min. After washing, cells were blocked overnight at 4°C in a humidified chamber. Cells were washed with cold DPBS and incubated with γH2A.X antibody (Cell Signaling, Cat. No. 9718s, 1:500) overnight at 4°C in a humidified chamber. After washing, cells were incubated with anti-rabbit secondary antibody conjugated with Alexa 568 (1 : 400, Life Technologies) and DAPI (Sigma Aldrich). Cells were captured with Nikon ECLIPSE Ti-S and Intensilight C-HGFI using a 10x lens.

Experiment result

1. Changes in DNA methylation during the acquisition of resistance to ceritinib

To explore DNA methylome and transcriptome changes during the course of acquisition of resistance to ALK inhibitors in NSCLC, we present an EML4-ALK fusion-positive lung cancer cell line, H3122, and previously established ceritinib. - 450K BeadChip DNA methylation analysis of resistant cell line (LR), RNA-seq and scRNA-seq were performed (FIG. 1A). Of the genes detectable by RNA-seq (n = 10,519), 13% genes (n = 1,403) were upregulated and 14% genes (n = 1,535) were downregulated in resistant cells (Fold Change > 2, FDR) < 0.05) (Fig. 1B). CYP4F11 (Cytochrome P450 4F11), AXL receptor tyrosine kinase (AXL), and EDIL3 (EGF like repeats and Discoidin Domains 3) were the most upregulated, and GLB1L2 (Galactosidase Beta 1 Like 2) and DUSP6 (Dual Specificity Phosphatase 6) were the most upregulated. has been significantly lowered. DNA methyltransferase (DNMT) and DNA demethylase (TET) were downregulated in resistant cells.

The present inventors confirmed the mRNA expression levels of DNMT and TET using qRT-PCR, confirming that not only transcriptome changes but also DNA methylome changes can occur during the process of resistance formation (Fig. 1C).

To investigate whether these transcriptional changes are associated with DNA methylation changes, we analyzed genome-wide methylation changes using the Infinium human methylation 450K array. In LR cells, 23,426 CpGs were hypermethylated and 17,797 CpGs were hypomethylated ( FIG. 1D ). Through integrated analysis of DNA methylation and gene expression, we found that 1,002 genes, including XYLT1 (Xylosyltransferase 1) and DUSP6, were hypermethylated and downregulated, and 809 genes, including Ankyrin Repeat Domain 2 (ANKRD2) and Cytidine deaminase (CDA), were It was confirmed that the canine genes were hypomethylated and up-regulated (Fig. 1E).

Hypermethylated and downregulated genes were associated with negative regulation of apoptosis, cell proliferation and MAPK signaling (Figure 1F), while hypomethylated and upregulated genes were associated with cell adhesion, cell migration and hippo signaling. (Fig. 1F). These data suggest that transcriptional plasticity can induce stable epigenetic modifications such as DNA methylation changes during the acquisition of ceritinib resistance.

2. Demethylation of CDA during ceritinib resistance acquisition

The expression level of CDA was increased 5-fold in the LR cell line ( FIG. 2C ). The promoter and putative enhancer regions of CDA showed reduced methylation in the LR cell line ( FIG. 2C ). These data suggest that transcriptional plasticity can induce DNA methylation changes in promoters and enhancers during the process of resistance acquisition.

3. Heterogeneity of naive and resistant cell lines

To explore the heterogeneity of naive and resistant cells, we performed scRNA-seq using 10X Genomics Single Cell 3' Solution. About 89,000 average reads/cell and 3,800 intermediate genes/cell were obtained from 7,532 cells (results not shown). According to t-SNE analysis, naive cells could be divided into 7 groups and resistant cells had one main group and two outlier groups (Fig. 3A and B).

Clusters 0, 4 and 7 had mostly resistant cells ( FIG. 3C ). Interestingly, these clusters showed G2/M and S phases in cell cycle analysis (Fig. 3D), indicating that resistant cells are highly proliferating. Most of the cells in clusters 3 and 9 were naïve, but also proliferated in G2/M or S stage. Trajectory analysis of all cells using monocles shows discrepancies between naive and resistant cells ( FIG. 3E ).

We detected 176 and 127 genes that exhibited 1.5-fold or more increased or decreased expression, respectively (FDR <0.001, Fig. 3F). Ferritin Light Chain (FTL), Cisplatin Resistance-Related Protein 9 (CLPTM1L), and CDA were the most upregulated genes in resistant cells.

4. Increased expression of CDA during ceritinib resistance formation

Next, cell-to-cell differences in CDA expression levels were identified using t-SNE analysis. As expected, CDA was highly expressed in resistant cell lines ( FIG. 4A ). Interestingly, we found CDA overexpressing cells among naive cells, suggesting that CDA overexpressing cells may have an advantage for ceritinib treatment.

In the violin plot result, it was also confirmed that CDA-overexpressing cell lines were mainly distributed in resistant cell clusters 0, 4, and 7, but it was confirmed that CDA-overexpressing cell lines were also rarely present in naive cell clusters ( FIG. 4B ).

Pseudotime-aligned expression heatmaps showed that CDA was continuously increased during the formation of ceritinib resistance (Figure 4C).

These data suggest that among naive cells, CDA overexpressing cells can be selected and propagated during the process of acquiring resistance.

5. 5hmdC and 5fdC selectively inhibit the growth of CDA overexpressing cells

CDA is one of the highest confidence candidates showing a consistent expression pattern between naive and resistant cells.

The present inventors investigated whether treatment with 5hmdC or 5fdC inhibited the growth of LR cells. Lung cancer cell lines H1299 and H1703 were used as controls with low CDA expression levels ( FIG. 5A ). Treatment with 5hmdC or 5fdC dose-dependently decreased colony formation in LR cells ( FIG. 5B ).

High dose (10 uM) 5hmdC or 5fdC treatment also reduced colony formation in H3122 but not H1299 and H1703. This suggests that 5hmdC or 5fdC selectively inhibits cell growth of CDA-overexpressing cells.

A similar dose-dependent effect was observed in the cell imaging assay (Fig. 5C). To determine whether 5fdC induces DNA damage in LR cells, we performed immunofluorescence staining for γ-H2AX in cells treated with 10 uM 5fdC. LR cells showed a 3-fold decrease in the number of proliferating cells and a 2.5-fold increase in the number of cells due to DNA damage ( FIG. 6 ). These results demonstrate that CDA overexpression resistant cells are vulnerable to 5fdC due to the accumulation of DNA damage.

6. Overexpression of CDA is associated with anticancer drug resistance and low survival rate of lung cancer patients

To investigate the clinical relevance of CDA in lung cancer, we analyzed CDA expression levels in lung adenocarcinoma (LUAD) using the TCGA and GTEx data-based tool, GEPIA. Although median expression levels of CDA were similar between tumors (n = 483, obtained from TCGA) and normal (n = 347, obtained from GTEx), one quarter of tumor samples had high expression levels of CDA compared to normal tissue samples. was shown (Fig. 7A). Furthermore, the overall survival (OS) of LUAD patients with high CDA expression was significantly lower than that of patients with low CDA expression (Fig. 7B).

According to the composition and method provided by the present invention, it is possible to accurately distinguish EML4-ALK-positive non-small cell lung cancer patients who are resistant to ALK inhibitors. can In addition, the composition of the present invention comprising a deoxycytidine analog can exhibit excellent anticancer effects in EML4-ALK-positive non-small cell lung cancer patients who are resistant to ALK inhibitors, and thus have very high industrial applicability.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for providing the information for selecting the drugs for treating EML4-ALK positive non-small cell lung cancer and compositions for treating non-small cell lung cancer resistant to ALK inhibitors <130> NP19-0138 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 146 <212> PRT <213> Artificial Sequence <220> <223> cytidine deaminase protein <400> 1 Met Ala Gln Lys Arg Pro Ala Cys Thr Leu Lys Pro Glu Cys Val Gln 1 5 10 15 Gln Leu Leu Val Cys Ser Gln Glu Ala Lys Lys Ser Ala Tyr Cys Pro 20 25 30 Tyr Ser His Phe Pro Val Gly Ala Ala Leu Leu Thr Gln Glu Gly Arg 35 40 45 Ile Phe Lys Gly Cys Asn Ile Glu Asn Ala Cys Tyr Pro Leu Gly Ile 50 55 60 Cys Ala Glu Arg Thr Ala Ile Gln Lys Ala Val Ser Glu Gly Tyr Lys 65 70 75 80 Asp Phe Arg Ala Ile Ala Ile Ala Ser Asp Met Gln Asp Asp Phe Ile 85 90 95 Ser Pro Cys Gly Ala Cys Arg Gln Val Met Arg Glu Phe Gly Thr Asn 100 105 110 Trp Pro Val Tyr Met Thr Lys Pro Asp Gly Thr Tyr Ile Val Met Thr 115 120 125 Val Gln Glu Leu Leu Pro Ser Ser Phe Gly Pro Glu Asp Leu Gln Lys 130 135 140 Thr Gln 145 <210> 2 <211> 441 <212> DNA <213> Artificial Sequence <220> <223> cytidine deaminase polynucleotide <400> 2 atggcccaga agcgtcctgc ctgcaccctg aagcctgagt gtgtccagca gctgctggtt 60 tgctcccagg aggccaagaa gtcagcctac tgcccctaca gtcactttcc tgtgggggct 120 gccctgctca cccaggaggg gagaatcttc aaagggtgca acatagaaaa tgcctgctac 180 ccgctgggca tctgtgctga acggaccgct atccagaagg ccgtctcaga agggtacaag 240 gatttcaggg caattgctat cgccagtgac atgcaagatg attttatctc tccatgtggg 300 gcctgcaggc aagtcatgag agagtttggc accaactggc ccgtgtacat gaccaagccg 360 gatggtacgt atattgtcat gacggtccag gagctgctgc cctcctcctt tgggcctgag 420 gacctgcaga agacccagtg a 441

Claims (16)

A composition for providing information for selecting a treatment drug for EML4-ALK-positive non-small cell lung cancer patients, comprising an agent for measuring the expression level of a cytidine deaminase (CDA) protein or gene, or a methylation level of a CDA gene, the treatment drug comprising:
1) ceritinib or crizotinib;
2) a deoxycytidine analog or a pharmaceutically acceptable salt thereof;
3) a combination of 1) and 2);
It is one selected from, and the deoxycytidine analog is 5hmdC (5-hydroxymethyl-2'-deoxycytidine), 5fdC (5-formyl-2'-deoxycytidine) or 5cadC (5-carboxyl-2'-deoxycitidine) A composition for providing information.
According to claim 1, wherein the agent is an antibody or aptamer that specifically binds to a CDA protein; or a primer or probe that specifically binds to the CDA gene.
The composition of claim 1, wherein the methylation level of the CDA gene is a methylation level at a promoter or enhancer region.
The method of claim 1, wherein the agent for measuring the CDA gene methylation level comprises a compound or methylation sensitive enzyme that modifies an unmethylated cytosine base, a primer specific for the methylated sequence of the CDA gene, and a primer specific for the unmethylated sequence. A composition comprising:
In order to provide information for drug selection for EML4-ALK-positive non-small cell lung cancer patients, expression levels of CDA (cytidine deaminase) proteins or genes, or CDA genes from biological samples provided from EML4-ALK-positive non-small cell lung cancer patients. An information providing method for selecting a treatment drug for a patient with EML4-ALK positive non-small cell lung cancer, comprising measuring the methylation level, wherein the treatment drug comprises:
1) ceritinib or crizotinib;
2) a deoxycytidine analog or a pharmaceutically acceptable salt thereof;
3) a combination of 1) and 2);
It is one selected from, and the deoxycytidine analog is 5hmdC (5-hydroxymethyl-2'-deoxycytidine), 5fdC (5-formyl-2'-deoxycytidine) or 5cadC (5-carboxyl-2'-deoxycitidine) How to provide information.
The method according to claim 5, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, lung tissue, cancer tissue, cervical or vaginal secretion, urine and cerebrospinal fluid. A method, characterized in that the sample.
The method according to claim 5, wherein the protein expression level is measured by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchteroni immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, FACS and A method characterized in that it is performed by a method selected from the group consisting of protein chips.
The method of claim 5, wherein the gene expression level measurement is performed by a method selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, and DNA chip. Way.
According to claim 5, wherein the methylation level measurement is PCR, methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), PCR using a methylated DNA-specific binding protein, quantitative PCR, DNA chip, A method characterized in that it is performed by a method selected from the group consisting of pyrosequencing and bisulfite sequencing.
The method of claim 5, wherein the expression level of the CDA (cytidine deaminase) protein or gene is higher than that of the control group, or the methylation level of the CDA gene is lower than that of the control group, ceritinib or crizotinib ), the method further comprising the step of determining that the treatment responsiveness of the non-small cell lung cancer patient to be low.
delete Prevention of EML4-ALK-positive non-small cell lung cancer that has acquired resistance to ceritinib or crizotinib, comprising a deoxycytidine analog or a pharmaceutically acceptable salt thereof as an active ingredient; or As a pharmaceutical composition for treatment, the deoxycytidine analog is 5hmdC (5-hydroxymethyl-2'-deoxycytidine), 5fdC (5-formyl-2'-deoxycytidine) or 5cadC (5-carboxyl-2'-deoxycitidine) Pharmaceutical composition, characterized in that.
The pharmaceutical composition according to claim 12, wherein the composition is administered in combination with an ALK inhibitor.
The pharmaceutical composition according to claim 12, wherein the composition and the ALK inhibitor are administered sequentially or separately as a single drug, or simultaneously administered in the form of a combination drug when the composition and the ALK inhibitor are administered in combination.
The method according to claim 12, wherein in the EML4-ALK-positive non-small cell lung cancer that has acquired resistance to ceritinib or crizotinib, CDA protein or mRNA expression is increased, or CDA gene methylation level A pharmaceutical composition, characterized in that it is reduced.
Acquisition of resistance to ceritinib or crizotinib in patients with EML4-ALK-positive non-small cell lung cancer, including agents that measure the expression level of a cytidine deaminase (CDA) protein or gene, or the methylation level of a CDA gene A composition for providing information regarding the determination of whether or not.

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