Pharmaceutical composition containing ODC1 inhibitor as active ingredient for inhibiting anticancer drug tolerance, recovering anticancer drug responsiveness, or enhancing anticancer drug sensitivity

Abstract

The present invention relates to a pharmaceutical composition for inhibiting anticancer drug tolerance, recovering anticancer drug responsiveness, or enhancing anticancer drug sensitivity, the composition containing an ODC1 inhibitor as an active ingredient. More specifically, it was found that if ODC1 expression or activity is inhibited when inducing EGFR-targeting drug tolerance using a non-small cell lung cancer cell model overexpressing a kinase involved in EGFR-targeting drug tolerance induction and an animal model xenografted with same, the acquisition of EGFR-targeting drug tolerance caused by kinase overexpression is inhibited and the tolerance is alleviated, and thus drug responsiveness is recovered. Therefore, an agent that inhibits the ODC1 expression or activity can be effectively used as an effective ingredient in a pharmaceutical composition for inhibiting anticancer drug tolerance, recovering anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

Description

A pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, containing an ODC1 inhibitor as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, containing an ODC1 inhibitor as an active ingredient.

[0002] Epithelial cancers, including prostate, breast, rectal, lung, pancreatic, ovarian, spleen, testicular, and thyroid cancers, are characterized by the abnormal, accelerated growth of epithelial cells. This accelerated growth initially leads to tumor formation and can eventually metastasize to other organs. Despite advances in the diagnosis and treatment of various cancers, these diseases still carry a significant mortality rate.

[0003] The epidermal growth factor receptor (EGFR) is a 170-kDa membrane-bound protein expressed on the surface of epithelial cells. EGFR is a member of the growth factor receptor family of protein tyrosine kinases, a class of cell cycle-regulating molecules. EGFR is activated when its ligand (EGF or TFRα) binds to its extracellular domain, resulting in autophosphorylation of the intracellular tyrosine kinase domain of the receptor.

[0004] EGFR is the protein product of the growth-promoting oncogene erbB or ErbB1, a member of the ERBB family of proto-oncogenes, and is thought to play a central role in the initiation and progression of many human cancers. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck, and stomach cancers, as well as glioblastoma. The ERBB family of oncogenes encodes four structurally related transmembrane receptors: EGFR, HER2 / neu (erbB2), HER-3 (erbB3), and HER-4 (erbB4). Clinically, ERBB oncogene amplification and / or receptor overexpression in tumors has been reported to correlate with disease recurrence and poor patient prognosis, as well as responsiveness to treatment.

[0005] Accordingly, EGFR-targeted therapies targeting EGFR are being developed as anticancer agents and applied clinically. For example, first-generation EGFR-targeted therapies such as AstraZeneca's Iressa (ingredient name: gefitinib) and Roche's Tarceva (ingredient name: erlotinib), second-generation EGFR-targeted therapies such as Pfizer's Vizimpro (ingredient name: dacomitinib) and Boehringer Ingelheim's Giotrif (ingredient name: afatinib), and third-generation EGFR-targeted therapies such as AstraZeneca's Tagrisso (ingredient name: osimertinib) are being prescribed as treatments for non-small cell lung cancer. However, it is known that most patients, including those who showed dramatic responses to EGFR-targeted therapies, eventually relapse. While numerous anticancer drugs targeting EGFR have been developed or are in development, their limitations include the development of resistance to their therapeutic effects after initial response, or the inability to respond at all. This represents a significant challenge for EGFR-targeted therapies, and suggests the need for research into drugs that suppress and / or eliminate acquired resistance.

[0006] Meanwhile, polyamines are polycationic alkylamines commonly found in all living cells, the most common examples being putrescine or spermine. Due to their flexible charge distribution, polyamines can bind to a variety of negatively charged macromolecules, including DNA, RNA, proteins, and acidic phospholipids. Therefore, they play a crucial role in cell growth, proliferation, differentiation, migration, gene regulation, and protein and nucleic acid synthesis, in addition to maintaining chromatin structure, regulating ion channels, maintaining membrane stability, and scavenging free radicals. In particular, increased intracellular polyamine concentrations are known to be associated with cell proliferation and tumorigenesis. For example, polyamine metabolism is often dysregulated in cancer. Furthermore, the polyamine pathway is a downstream target of many oncogenes.

[0007] Accordingly, the present inventors have made efforts to develop a new approach for cancer treatment in individuals that no longer respond to such EGFR-targeted anticancer drug treatment, and a therapeutic agent to solve this problem. As a result, we created a cell model for screening genes involved in inducing EGFR-targeted drug resistance using cancer cell lines that show EGFR-targeted drug sensitivity, and after overexpressing 589 kinases in these cell lines and treating them with EGFR-targeted drugs, we screened and analyzed cells in which resistance was induced, thereby deriving kinases involved in inducing EGFR-targeted drug resistance. In addition, using the cell model overexpressing the derived kinases, we confirmed that the polyamine pathway involving MYC and ODC1 plays an important role when resistance to EGFR-targeted drugs is acquired, and that ODC1 is regulated by MYC in this pathway. Accordingly, by suppressing ODC1 expression or activity in EGFR-targeted therapy resistance-induced cell models and animal models and regulating the polyamine pathway, we confirmed the effect of suppressing acquired resistance to EGFR-targeted therapy caused by kinase overexpression and restoring drug responsiveness. Accordingly, we have revealed that the agent that suppresses ODC1 expression or activity can be usefully utilized as an active ingredient in a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, leading to the present application.

[0008] [Prior Art Literature]

[0009] [Non-patent literature]

[0010] Gullick W, Marsden J, Whittle N, et al: Expression of epidermal growth factor receptors on human cervical, ovarian, and vulval carcinomas. Cancer Res 1986;46:285-292

[0011] S Cohenet al: Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. J Biol Chem 1980;255:4834-4842

[0012] A. B. Schreiberet al: Biological Role of Epidermal Growth Factor-Receptor Clusterin. J. Biol. Chem 1983 258;846-853

[0013] L. Harriset al: C-erbB-2 in serum of patients with breast cancer. Int J Biol Markers 1999;14:8-15

[0014] J. Mendelsohn and J. Baselga: The EGF receptor family as targets for cancer therapy. Oncogene 2000;19(56):6550-6565

[0015] C. R. Carlinet al: Biosynthesis and glycosylation of the epidermal growth factor receptor in human tumor-derived cell lines A431 and Hep 3B. Mol. Cell. Biol 1986; 6:257-264

[0016] Kye Young Lee: Targeted Therapy of Lung Cancer. J Korean Med Assoc 2008;51(5):483-491

[0017] J. Li et al: Polyamines and related signaling pathways in cancer. Cancer Cell International 2020;20:539

[0018] Conventional EGFR-targeting anticancer drugs initially show therapeutic effects, but long-term administration leads to resistance, resulting in ineffective treatment. Therefore, new approaches are needed to address the problem of acquired resistance in the use of these EGFR-targeting anticancer drugs.

[0019] The purpose of the present invention is to provide a composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, which contains an ODC1 inhibitor as an active ingredient as a new approach.

[0020] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity; a use of an ODC1 inhibitor for preparing a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity; and a method for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, comprising administering an ODC1 inhibitor to a subject.

[0021] In addition, the present invention provides a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer; a use of an ODC1 inhibitor for preparing a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer; and a method for preventing or treating anticancer drug-resistant cancer, comprising administering an ODC1 inhibitor to a subject.

[0022] In addition, the present invention provides an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer; a use of an ODC1 inhibitor for manufacturing an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer; and a method for preventing or treating anticancer drug-resistant cancer, comprising administering an ODC1 inhibitor and an anticancer agent to a subject.

[0023] In addition, the present invention

[0024] 1) a step of measuring the expression level of one or more genes or proteins thereof from a sample isolated from a cancer patient, including CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK; and

[0025] 2) A method for providing information for predicting response and prognosis to an anticancer drug is provided, including a step of determining that anticancer drug resistance will be suppressed by co-administering an anticancer drug and an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample.

[0026] In addition, the present invention

[0027] 1) A step of measuring the expression level of one or more genes or proteins thereof among CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK from a sample isolated from a patient with anticancer drug-resistant cancer; and

[0028] 2) A method for providing information for predicting response and prognosis to an anticancer drug is provided, including a step of determining that anticancer drug sensitivity will be restored by administering an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample.

[0029] The present inventors have confirmed that when resistance to EGFR-targeted therapy is acquired through kinase overexpression in cancer, the polyamine pathway involving MYC and ODC1 plays a significant role, and that ODC1 is regulated by MYC in this pathway. Accordingly, by suppressing ODC1 expression or activity in cell models and animal models of EGFR-targeted therapy resistance induction and thereby regulating the polyamine pathway, the acquired resistance to EGFR-targeted therapy due to kinase overexpression was suppressed and drug responsiveness was restored. Therefore, agents that suppress ODC1 expression or activity can be usefully utilized as active ingredients in pharmaceutical compositions for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

[0030] Figure 1 is a schematic diagram illustrating the process of producing a cell model for screening genes involved in inducing EGFR target therapy resistance.

[0031] Figure 2 is a diagram confirming genes involved in inducing resistance to Erlotinib, a first-generation EGFR targeted therapy.

[0032] Figure 3 is a diagram showing genes involved in inducing resistance to erlotinib, a first-generation EGFR targeted therapy, and / or osimertinib, a third-generation EGFR targeted therapy.

[0033] Figure 4 is a diagram showing the drug response after treating erlotinib at various concentrations in 10 kinase-overexpressing cell models that recovered drug response to erlotinib after a drug holiday period.

[0034] Figure 5 is a diagram showing whether 10 kinase-overexpressing cell models that recovered drug responsiveness to erlotinib after a drug holiday period acquired re-resistance to erlotinib.

[0035] Figure 6 is a diagram illustrating the pathways regulated according to erlotinib responsiveness as revealed by signaling pathway system analysis (GESA) based on data obtained through RNA profiling in AXL, ALK or FES overexpressing cell models that recovered drug responsiveness to erlotinib after a drug holiday period.

[0036] Figure 7 is a diagram confirming the increase in polyamine signaling pathways as shown by signaling pathway system analysis (GESA) based on data obtained through RNA profiling in cell models overexpressing six kinases (CRKL, SYK, AXL, ERBB2, FES, or ALK) in which erlotinib resistance was induced.

[0037] Figure 8 is a ranking plot of genes that do not acquire re-resistance to erlotinib after introduction of pooled shRNA screening in an ALK overexpressing cell model that recovered drug responsiveness to erlotinib after a drug holiday period.

[0038] Figure 9 is a diagram confirming the increase in MYC and ODC1 expression due to induction of erlotinib resistance in five kinase overexpressing cell models (AXL, ALK, CSF1R, FES, YES1) that recovered drug responsiveness to erlotinib after a drug holiday period.

[0039] Figure 10 is a diagram confirming the inhibitory effect of acquisition of erlotinib resistance according to the presence or absence of ODC1 inhibition using shRNA in a cell model overexpressing FGFR1 or ERBB2.

[0040] Figure 11 is a diagram showing the inhibitory effect of acquisition of erlotinib resistance according to the presence or absence of MYC inhibition using shRNA in 10 kinase overexpressing cell models that recovered drug responsiveness to erlotinib after a drug holiday period.

[0041] Figure 12 is a diagram confirming the effect of restoring responsiveness to erlotinib according to the presence or absence of ODC1 inhibition using shRNA in 29 kinase overexpression cell models in which erlotinib resistance was induced.

[0042] Figure 13 is a diagram confirming the effect of restoring responsiveness to erlotinib according to the presence or absence of MYC inhibition using shRNA in 11 kinase overexpression cell models in which erlotinib resistance was induced.

[0043] Figure 14 is a diagram confirming the effect of reducing ODC1 protein expression by MYC inhibition using shRNA in seven kinase overexpression cell models in which erlotinib resistance was induced.

[0044] Figure 15 is a diagram confirming the effect of restoring responsiveness to osimertinib according to the presence or absence of ODC1 inhibition using shRNA in 16 kinase overexpression cell models in which osimertinib resistance was induced.

[0045] Figure 16 is a diagram confirming the effect of restoring responsiveness to osimertinib according to the presence or absence of MYC inhibition using shRNA in 16 kinase overexpression cell models in which osimertinib resistance was induced.

[0046] Figure 17 is a diagram confirming the effect of reducing ODC1 protein expression by MYC inhibition using shRNA in 16 kinase overexpression cell models in which osimertinib resistance was induced.

[0047] Figure 18 is a diagram confirming the apoptosis effect according to the presence or absence of ODC1 inhibition using shRNA in 19 kinase overexpression cell models in which erlotinib resistance was induced.

[0048] Figure 19 is a diagram confirming the cell death effect according to the presence or absence of ODC1 inhibition using shRNA in 16 types of kinase overexpression cell models in which osimertinib resistance was induced.

[0049] Figure 20 is a diagram confirming the inhibitory effect of acquisition of erlotinib resistance after co-treatment with erlotinib and DMFO, an ODC1 activity inhibitor, in a cell model overexpressing FGFR1 or ERBB2.

[0050] Figure 21 is a diagram showing the recovery of responsiveness to erlotinib after co-treatment with erlotinib and DMFO, an ODC1 activity inhibitor, in 28 kinase overexpression cell models in which erlotinib resistance was induced.

[0051] Figure 22 is a diagram showing the recovery of responsiveness to osimertinib after co-treatment with osimertinib and DMFO, an ODC1 activity inhibitor, in 16 kinase overexpression cell models in which osimertinib resistance was induced.

[0052] Figure 23 shows the recovery of responsiveness to lazertinib after co-treatment with lazertinib and DMFO, an ODC1 activity inhibitor, in five kinase overexpression cell models in which osimertinib resistance was induced.

[0053] Figure 24 is a diagram showing the inhibitory effect on acquisition of erlotinib resistance after co-administration of erlotinib and DMFO, an ODC1 activity inhibitor, in a mouse model transplanted with FGFR1 or ERBB2 overexpressing cells.

[0054] Figure 25 is a diagram showing the recovery effect of responsiveness to erlotinib after co-administration of erlotinib and DMFO, an ODC1 activity inhibitor, in a mouse model transplanted with erlotinib-resistant cells overexpressing FGFR1 or ERBB2.

[0055] Figure 26 is a diagram showing the recovery effect of responsiveness to osimertinib after co-administration of osimertinib and DMFO, an ODC1 activity inhibitor, in a mouse model transplanted with YES1 or AXL overexpressing osimertinib-resistant inducing cells.

[0056] Figure 27 is a diagram confirming ODC1 expression in tumor tissues that acquired resistance through long-term administration of erlotinib to a mouse model transplanted with erlotinib-resistant cells overexpressing FGFR1 or ERBB2.

[0057] Figure 28 is a diagram confirming increased ODC1 expression in tumor tissue of an erlotinib-resistant patient.

[0058] Hereinafter, the present invention will be described in detail.

[0059] The present invention is characterized in that it uses kinases and their genes as biomarkers to confirm whether there is resistance and re-resistance to EGFR targeted therapy as an anticancer agent, and uses a cell model that overexpresses the kinase involved in the resistance and re-resistance to identify the pathways that are key to controlling EGFR targeted therapy resistance and re-resistance and the genes involved in the pathways. Accordingly, the present invention confirmed that the MYC pathway and the polyamine pathway are important as the pathways, and that ODC1 and MYC, specifically ODC1, are important as factors involved in these pathways.

[0060] Accordingly, the present invention provides a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity; a use of an ODC1 inhibitor for preparing a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity; and a method for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, comprising administering an ODC1 inhibitor to a subject.

[0061] In the present invention, the ODC1 inhibitor may inhibit ODC1 expression or activity.

[0062] In addition, ODC1 inhibitors that suppress the ODC1 expression include, but are not limited to, antisense nucleotides that complementarily bind to the mRNA of the ODC1 gene, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or vectors containing them. Such antisense nucleotides, siRNA, shRNA, miRNA, or vectors containing them can be produced using methods known in the art.

[0063] The above "antisense nucleotides" are those that bind (hybridize) to complementary base sequences in DNA, immature mRNA, or mature mRNA, thereby disrupting the flow of genetic information from DNA to protein, as defined by Watson-Crick base pairing. The specificity of antisense nucleotides for their target sequence makes them exceptionally versatile. Because antisense nucleotides are long chains of monomeric units, they can be easily synthesized against target RNA sequences. Recent studies have demonstrated the utility of antisense nucleotides as a biochemical tool for studying target proteins (Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Recent advances in oligonucleotide chemistry and the synthesis of nucleotides exhibiting enhanced cell adhesion, target binding affinity, and nuclease resistance suggest that the use of antisense nucleotides can be considered a new type of inhibitor.

[0064] The above "siRNA" refers to a double-stranded RNA that induces RNA interference by cleaving the mRNA of a target gene, and is composed of an RNA strand of a sense sequence having the same sequence as the mRNA of the target gene and an RNA strand of an antisense sequence having a sequence complementary thereto. The above siRNA may include a form in which the siRNA itself is synthesized in vitro or a form in which the base sequence encoding the siRNA is inserted into an expression vector and expressed.

[0065] The above "shRNA" refers to a single-stranded, 50-60 nucleotide RNA that forms a stem-loop structure in vivo. In other words, shRNA is an RNA sequence that forms a tight hairpin structure to suppress gene expression through RNA interference (RNAi). A loop region of 5-10 nucleotides is complementarily paired with a long RNA of 15-30 nucleotides on either side to form a double-stranded stem. shRNA is typically transduced into cells via a vector containing the U6 promoter to ensure expression, and is usually passed on to daughter cells to ensure heritable gene suppression. The shRNA hairpin structure is cleaved by intracellular mechanisms into siRNA, which then binds to the RNA-induced silencing complex (RISC). RISC binds to and cleaves mRNA. shRNA is transcribed by RNA polymerase.

[0066] The above "miRNA" refers to a short non-coding RNA consisting of approximately 22 nucleotides. It is known to function as a post-transcriptional regulator during gene expression. By complementarily binding to target mRNAs with complementary nucleotide sequences, it degrades the target mRNAs or inhibits their translation into proteins.

[0067] The above vector refers to a genetic construct containing foreign DNA inserted into a genome encoding a polypeptide. The vector related to the present invention is a vector in which a nucleic acid sequence that inhibits the above gene is inserted into the genome, and examples of these vectors include DNA vectors, plasmid vectors, cosmid vectors, bacteriophage vectors, yeast vectors, or viral vectors.

[0068] In addition, ODC1 inhibitors that suppress the ODC1 activity include, but are not limited to, compounds, peptides, peptide mimetics, aptamers, antibodies, or natural products that specifically bind to the ODC1 protein.

[0069] The above peptide mimetics are peptides or non-peptides that inhibit the binding domain of the ODC1 protein that drives ODC1 activity. The major residues of non-hydrolyzable peptide analogs can be produced using a β-turn dipeptide core (Nagai et al. Tetrahedron Lett 26:647, 1985), keto-methylene pseudopeptides (Ewenson et al. J Med chem 29:295, 1986), azepines, benzodiazepines, β-aminoalcohols (Gordon et al. Biochem Biophys Res commun 126:419, 1985), and substituted gamma-lactam rings.

[0070] The above aptamer is a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and can bind to a target molecule with high affinity and specificity. Since the first development of the aptamer discovery technology called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) (Ellington, AD and Szostak, JW., Nature 346:818-822, 1990), many aptamers that can bind to various target molecules, including small-molecule organic compounds, peptides, and membrane proteins, have continued to be discovered. Because of their unique characteristics of being able to bind to target molecules with high affinity (usually pM level) and specificity, aptamers are comparable to single antibodies, and in particular, they have high potential as a substitute for antibodies, to the point where they are called "chemical antibodies."

[0071] The above compounds may be low molecular weight therapeutic compounds, for example, compounds weighing about 1000 Da, such as 400 Da, 600 Da, or 800 Da. Depending on the purpose, such compounds may form part of a compound library, the number of compounds constituting the library also varying from tens to millions. Such compound libraries may include, but are not limited to, peptides and other cyclic or linear oligomeric compounds, and template-based low molecular weight compounds such as benzodiazepines, hydantoins, biaryls, carbocyclic and polycyclic compounds (e.g., naphthalenes, phenothiazines, acridines, steroids, etc.), carbohydrates and amino acid derivatives, dihydropyridines, benzhydryls, and heterocycles (e.g., triazines, indoles, thiazolidines, etc.), which are merely exemplary.

[0072] Specifically, compounds that specifically bind to the ODC1 protein include, but are not limited to, DFMO.

[0073] In the present invention, the ODC1 inhibitor can additionally suppress or alleviate the acquisition of resistance due to anticancer drug administration after an anticancer drug holiday.

[0074] In the present invention, the anticancer agent may be an EGFR (Epidermal Growth Factor Receptor) targeted therapeutic agent, and examples of the EGFR targeted therapeutic agent include erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib, pelitinib, neratinib, icotinib, brigatinib, lapatinib, canertinib, vandetanib, PKI-166 or AEE788, and specifically, erlotinib or osimertinib.

[0075] As used herein, the term "resistance" or "tolerance" refers to a state in which a patient is not sensitive to a drug and thus the drug does not work effectively. If resistance develops or is likely to develop after administration of the anticancer drug, specifically the EGFR-targeted therapy, administration of the EGFR-targeted therapy may be discontinued for a certain period of time. This "drug holiday" (or "drug holiday," "medical holiday," "systematic treatment interruption," "strategic treatment interruption," or "drug holiday") may reduce the risk of adverse (i.e., treatment-related) effects, restore sensitivity to the EGFR-targeted therapy and some normal physiological functions in the patient, or improve patient compliance. The timing of the drug holiday period may vary from patient to patient and may be selected based on the clinical evaluation of the treating physician, and may be, for example, 2 weeks to 6 months, 2 weeks to 5 months, 2 weeks to 4 months, 2 weeks to 12 weeks, 2 weeks to 11 weeks, 2 weeks to 10 weeks, 2 weeks to 9 weeks, 2 weeks to 8 weeks, 2 weeks to 7 weeks, 2 weeks to 6 weeks, 2 weeks to 5 weeks, 2 weeks to 4 weeks, or 2 weeks to 3 weeks. In addition, "retolerance" means that after completing the drug holiday period, the patient does not become sensitive to the drug again when the drug is re-administered, so that the drug does not work effectively.

[0076] In the present invention, the anticancer agent, specifically the EGFR targeted therapeutic agent, exhibits resistance in cancer, and examples of the cancer include breast cancer, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell carcinoma, thyroid cancer, stomach cancer, bladder cancer, or glioblastoma, and specifically, non-small cell lung cancer, but is not limited thereto.

[0077] In addition, the cancer may be a cancer in which the expression level of one or more genes or proteins thereof is high among CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK compared to a normal control.

[0078] Specifically, cancers with high expression levels of one or more genes or proteins thereof among CSF1R, RAF1, SRC, ALK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, CRKL, FRK, BLK and BTK compared to normal controls exhibit resistance to erlotinib, and the resistance is suppressed by an ODC1 inhibitor, drug responsiveness to erlotinib is restored, and drug sensitivity to erlotinib is enhanced. In addition, cancers with high expression levels of one or more genes or proteins thereof among CSF1R, ALK, ITK, AXL, PDGFRA, ARAF, FGR, FES, FGFR2, YES1, PIK3CG, CRKL, and FRK compared to normal controls exhibit resistance to osimertinib, and the resistance is suppressed by an ODC1 inhibitor, drug response to osimertinib is restored, and drug sensitivity to osimertinib is enhanced.

[0079] In a specific embodiment of the present invention, the inventors identified a kinase involved in inducing EGFR-targeted therapy resistance in non-small cell lung cancer, and confirmed that the polyamine pathway is important when resistance to EGFR-targeted therapy is acquired through kinase overexpression. Furthermore, ODC1 and MYC, which are involved in the polyamine pathway, play a significant role, and ODC1 is regulated by MYC in the pathway, confirming that OCD1 is a target for suppressing the induction of EGFR-targeted therapy resistance through kinase overexpression.

[0080] In addition, the present inventors used a cell model overexpressing a kinase involved in inducing EGFR-targeted therapy resistance in non-small cell lung cancer, an animal model xenografted with the cell model, and a cell model overexpressing the kinase and induced to develop resistance to erlotinib or osimertinib as an EGFR-targeted therapy, and confirmed that as a result of inhibiting ODC1 expression or activity, acquired resistance to EGFR-targeted therapy due to kinase overexpression was inhibited, and resistance was alleviated, resulting in an effect of restoring drug responsiveness to EGFR-targeted therapy.

[0081] In addition, the present inventors overexpressed a kinase involved in inducing EGFR-targeted therapy resistance and used a cell model in which erlotinib resistance was induced by an EGFR-targeted therapy or a cell model in which drug responsiveness was restored after a drug holiday period after inducing erlotinib resistance, and confirmed that the effect was the same as that by inhibiting ODC1 expression when MYC expression was inhibited.

[0082] Therefore, the present inventors confirmed that when resistance to EGFR targeted therapy is acquired by overexpression of kinases involved in inducing resistance to the therapeutic agent in cancer, specifically in non-small cell lung cancer, the polyamine pathway involving MYC and ODC1 plays an important role, and that ODC1 is regulated by MYC in the pathway. Accordingly, by suppressing ODC1 expression or activity in cell models and animal models of EGFR targeted therapy resistance induction, thereby regulating the polyamine pathway, the effects of suppressing acquired resistance to EGFR targeted therapy due to kinase overexpression, alleviating resistance, and restoring drug responsiveness were confirmed. Therefore, the agent that suppresses ODC1 expression or activity can be usefully used as an active ingredient of a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

[0083] In addition, the present invention provides a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer; a use of an ODC1 inhibitor for preparing a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer; and a method for preventing or treating anticancer drug-resistant cancer, comprising administering an ODC1 inhibitor to a subject.

[0084] Also provided are a pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, which further comprises an anticancer agent; a use of an ODC1 inhibitor for use in a pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, which further comprises an anticancer agent; and a use of an ODC1 inhibitor for preparing a pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, which further comprises an anticancer agent.

[0085] In addition, the present invention provides an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer, comprising an ODC1 inhibitor as an active ingredient; a use of an ODC1 inhibitor for use as an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer; a use of an ODC1 inhibitor for producing an anticancer adjuvant for preventing or treating anticancer drug-resistant cancer; and a method for preventing or treating anticancer drug-resistant cancer, comprising administering an ODC1 inhibitor and an anticancer agent to a subject.

[0086] In the present invention, the description of the inhibitor is as described above.

[0087] In the present invention, the anticancer agent may be an EGFR targeted therapeutic agent, and examples of the EGFR targeted therapeutic agent include erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib, felitinib, neratinib, icotinib, brigatinib, lapatinib, canertinib, vandetanib, PKI-166 or AEE788, and specifically, erlotinib or osimertinib.

[0088] In the present invention, the cancer may include breast cancer, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell carcinoma, thyroid cancer, stomach cancer, bladder cancer, or glioblastoma, and may specifically be non-small cell lung cancer, but is not limited thereto.

[0089] In addition, the cancer may be a cancer in which the expression level of one or more genes or proteins thereof is high among CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK compared to a normal control.

[0090] Specifically, cancers with high expression levels of one or more genes or proteins thereof among CSF1R, RAF1, SRC, ALK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, CRKL, FRK, BLK and BTK compared to normal controls may exhibit resistance to erlotinib, and the resistance may be suppressed by an ODC1 inhibitor, and drug responsiveness to erlotinib may be restored and drug sensitivity to erlotinib may be enhanced, thereby exhibiting a cancer treatment effect. In addition, cancers with high expression levels of one or more genes or proteins thereof among CSF1R, ALK, ITK, AXL, PDGFRA, ARAF, FGR, FES, FGFR2, YES1, PIK3CG, CRKL, and FRK compared to normal controls may exhibit resistance to osimertinib, and the resistance may be suppressed by an ODC1 inhibitor, and drug responsiveness to osimertinib may be restored and drug sensitivity to osimertinib may be enhanced, thereby exhibiting a cancer treatment effect.

[0091] The present inventors have confirmed the effect of suppressing ODC1 expression or activity when inducing EGFR-targeted therapy resistance by using a non-small cell lung cancer cell model overexpressing a kinase involved in inducing EGFR-targeted therapy resistance and an animal model xenografted with the same, thereby suppressing acquired resistance to EGFR-targeted therapy due to kinase overexpression and alleviating resistance to restore drug responsiveness. Therefore, the agent that suppresses ODC1 expression or activity can be usefully used as an active ingredient of a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer, or an anticancer adjuvant agent.

[0092] In the composition of the present invention, the inhibitor may be used in the form of a pharmaceutically acceptable salt, and as a salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyn-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, Contains terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0093] The acid addition salt according to the present invention can be prepared by conventional methods, for example, by dissolving the compound of the present invention in an excess aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, the salt can be prepared by evaporating the solvent or excess acid from the mixture to dryness, or by suction filtration of the precipitated salt.

[0094] Additionally, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are pharmaceutically suitable as metal salts. Furthermore, the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0095] The pharmaceutical composition of the present invention can be prepared in the form of a pharmaceutical composition for treating cancer, which additionally contains a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, wherein the carrier may include a non-naturally occurring carrier. Specifically, the pharmaceutical composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, each according to a conventional method.

[0096] Specific examples of carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc.

[0097] When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0098] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0099] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0100] Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0101] Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0102] The content of the inhibitor included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50 wt%, more specifically 0.01 to 20 wt%, based on the total weight of the final composition.

[0103] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field. The composition of the present invention may be administered as a compounded individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0104] The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng / kg to about 100 mg / kg, specifically about 1 ng / kg to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but can be administered once a day or administered in divided doses several times. The above dosage does not limit the scope of the present invention in any way.

[0105] In addition, the present invention

[0106] 1) a step of measuring the expression level of one or more genes or proteins thereof from a sample isolated from a cancer patient, including CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK; and

[0107] 2) A method for providing information for predicting response and prognosis to an anticancer drug is provided, including a step of determining that anticancer drug resistance will be suppressed by co-administering an anticancer drug and an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample.

[0108] In addition, the present invention

[0109] 1) A step of measuring the expression level of one or more genes or proteins thereof from a sample isolated from a patient with anticancer drug-resistant cancer, including CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK;

[0110] 2) A method for providing information for predicting response and prognosis to an anticancer drug is provided, including a step of determining that anticancer drug sensitivity will be restored by co-administering an anticancer drug and an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample.

[0111] In the method according to the present invention, the description of the inhibitor, anticancer drug resistance, and cancer is as described above.

[0112] In the method according to the present invention, the sample of step 1) may include, but is not limited to, tissue, cells, blood, serum, plasma, saliva, and urine.

[0113] In the method according to the present invention, the method for measuring the expression level of the gene or its protein can be performed by including a known process for isolating mRNA or protein from a sample using a known technique.

[0114] Measurement of the expression level of the above gene is specifically measuring the level of mRNA, and methods for measuring the level of mRNA include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction, RNase protection assay, Northern blot, and DNA chip.

[0115] The measurement of the above protein level can utilize antibodies. In this case, the protein in the sample and the antibody specific therefor form a complex, i.e., an antigen-antibody complex, and the amount of the antigen-antibody complex formed can be quantitatively measured through the size of the signal of the detection label. Such detection labels can be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes, but are not limited thereto. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, orchite immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, etc.

[0116] The present inventors have confirmed the effect of suppressing kinase overexpression and alleviating resistance by suppressing ODC1 expression or activity when inducing EGFR targeted therapy resistance using a non-small cell lung cancer cell model overexpressing a kinase involved in inducing EGFR targeted therapy resistance and an animal model xenografted with the same, thereby suppressing acquired resistance to EGFR targeted therapy due to kinase overexpression and restoring drug responsiveness by alleviating resistance. Therefore, the method for measuring kinase overexpression can be used to predict the response to and prognosis of an anticancer drug after co-administration of an anticancer drug and an agent that suppresses ODC1 expression or activity.

[0117] Hereinafter, the present invention will be described in detail by examples.

[0118] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0119] <Example 1> Acquisition of cells incapable of inducing resistance to EGFR targeted therapy

[0120] As shown in the schematic diagram in Fig. 1, monoclonal cells without the EGFR T790M mutation were isolated from cancer cell lines showing sensitivity to EGFR targeted therapy, and drug resistance was induced in the isolated monoclonal cells by long-term treatment with EGFR targeted therapy. As a result, three types of monoclonal cells were obtained: specifically, monoclonal cells incapable of inducing resistance, monoclonal cells in which resistance to erlotinib was induced by a new occurrence of the EGFR T790M mutation (T790M-bearing resistant clones), and monoclonal cells in which resistance was induced without the EGFR T790M mutation (non-T790M-bearing resistant clones).

[0121] <1-1> Isolation of single clone cells from cancer cell lines showing sensitivity to EGFR targeted therapy

[0122] PC9 cells, a non-small cell lung cancer (NSCLC) cell line, harbor the EGFR del19 mutation. They exhibit short-term responsiveness to the first-generation EGFR-targeted therapy, erlotinib, but are known to develop resistance after long-term treatment of six months to one year. Therefore, a single clone was isolated from PC9 cells to develop a cancer cell line exhibiting sensitivity to EGFR-targeted therapy.

[0123] Specifically, PC9 cells were cultured in a cell incubator at 5% CO2 and 37°C using RPMI-1640 medium (Corning, USA) supplemented with 10% fetal bovine serum (FBS, Merck, USA) and 1% antibiotics (Invitrogen, USA). For single cell isolation, 10 ml of a solution containing RPMI-1640 medium, 10% FBS, 1% antibiotics (penicillin-streptomycin, RPMI 10%), and 0.5% Select agar was added to a 100 mm cell culture dish and solidified at room temperature for 30 minutes. After that, 1 × 10 PC9 cells were cultured.3 A 5 ml solution containing 10% RPMI-1640 medium and 0.4% Select agar was dispensed onto the dish and allowed to solidify at room temperature for 15 minutes. After confirming that single cells had separated from the dish, they were cultured in a cell incubator at 5% CO2 and 37°C.

[0124] After 2-3 weeks, single cell lines with formed colonies were picked up using a 200 μl tip and dispensed into 96-well plates containing 100 μl of culture solution (RPMI-1640 medium, 10% FBS, 1% PS mixture). Then, at intervals of 5-7 days, when the cell density in each well plate reached 80%, the cells were sequentially transferred to larger plates such as 24-well plates, 12-well plates, and 6-well plates to obtain single clone cells.

[0125] In addition, Sanger sequencing was performed on the single clone cells to confirm the presence of the EGFR T790M mutation, thereby obtaining a total of 30 single clone cells that did not have the EGFR T790M mutation, which is well known as a conventional resistance-inducing mechanism.

[0126] <1-2> Selection of monoclonal cells incapable of inducing EGFR-targeted therapy resistance in monoclonal cells

[0127] Each of the 30 single clone cells obtained in the above Example <1-1> was seeded at 5×10 in a 96-well plate. 3 Cells were seeded at 1 μM per well and treated with 1 μM erlotinib. The drug was replaced with a new one every 3-4 days, and cell proliferation was observed to determine whether resistance was induced. When erlotinib responsiveness was no longer observed and cell proliferation was confirmed, the cells were sequentially transferred to a larger plate and Sanger sequencing was performed using the same method described in Example <1-1> to determine whether the EGFR T790M mutation was present.

[0128] Through this, as shown in Figure 1, we identified and isolated monoclonal cells that were incapable of inducing resistance at all, monoclonal cells in which resistance was induced by the EGFR T790M mutation, and monoclonal cells in which resistance was induced by causes other than the EGFR T790M mutation. Specifically, the point at which responsiveness to erlotinib was no longer observed and cell proliferation was confirmed was approximately 6 months after erlotinib treatment, and at this time, 16 monoclonal cells in which erlotinib resistance was induced were identified. Of these, 7 types (non-T790M-bearing resistant clones) were confirmed to have induced resistance without the EGFR T790M mutation. In other words, it was confirmed that resistance was induced by causes other than the EGFR T790M mutation. In addition, it was confirmed that the remaining 9 types (T790M-bearing resistant clones) were induced to develop resistance by the newly occurring EGFR T790M mutation, which is a well-known mutation.

[0129] Additionally, 14 types of single clone cells (Resistance refractory clones) were identified as resistance-inducible single clone cells that do not induce erlotinib resistance, and these were used to screen for genes involved in inducing resistance to EGFR targeting therapeutics.

[0130] <Example 2> Production of a cell model for inducing resistance to first-generation EGFR targeted therapy.

[0131] If drug resistance is induced by overexpression of a kinase in cells with a genetic background that does not induce drug resistance at all, it can be assumed that the overexpressed kinase is involved in the induction of drug resistance. Accordingly, the kinase was overexpressed in the monoclonal cells incapable of inducing resistance obtained in <Example 1> above, and after treatment with the first-generation EGFR targeted therapy, Erlotinib (ER), cells in which resistance was induced were confirmed.

[0132] Specifically, a 589-ORF library (CCSB / Broad Institute Kinase ORF collection) was used for kinase overexpression. The 589-ORF library was introduced into the PLX302-V5 (addgene, 25896) vector via LR-reaction (invitrogen, USA). Viruses were constructed to transfect cells with the obtained vector. The virus was seeded on 293T cells (culture medium condition = DMEM medium, 10% FBS, 1% PS) in a 6-well cell culture dish (Corning, USA), and after 24 hours, when the cell density reached 30%, 0.5 μg of pMD2.G (addgene, 12259), 0.5 μg of psPAX2 (addgene, 12260), 1 μg of ORF DNA (each of 589 ORF library introduction vectors), 6 μl of Fugene (Promega), and 100 μl of optimem (Gibco) were mixed to produce 589 viruses. In order to transfect the virus, 2 types (PC9-1 and PC9-6) were randomly selected among the 14 types of resistance-inducing monoclonal cells obtained in the above <Example 1> and seeded in a 96-well plate at 5 × 10 3 Cells were seeded at 10 cells / well, and the culture medium was removed after 24 hours. Then, 70 μl of the virus and 10 μg / ml of polybrene were added, and the cells were cultured for 12 hours in a cell incubator at 5% CO2 and 37°C. After 12 hours, the virus was removed, and 100 μl of RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics was added. After 48 hours, 2 μg / ml of puromycin was treated for 72 hours to select only virus-infected cells, and 1,200 PC9-1 and PC9-6 cells overexpressing each of the 589 kinases were obtained.

[0133] Afterwards, the 1,200 cells were seeded into 96-well plates, and when the cell density reached 80%, each cell was treated with 1 μM erlotinib. After 48 hours, the culture medium was replaced with fresh erlotinib, and the cells were treated with 1 μM erlotinib. Thereafter, the culture medium and erlotinib were replaced with fresh ones at 7-day intervals. Cell proliferation was observed before replacing with the new drug to determine whether resistance was induced.

[0134] As a result, as shown in Fig. 2, 29 kinases and their genes that repeatedly appeared among 589 kinases were identified in PC9-1 and PC9-6 cells in which erlotinib resistance was induced.

[0135] The above results demonstrate that the 29 kinases are involved in inducing erlotinib resistance. Therefore, the cell line overexpressing these kinases was used as a cell model system for screening drugs that suppress resistance to EGFR-targeted anticancer agents.

[0136] <Example 3> Production of a cell model for inducing resistance to third-generation EGFR targeted therapy.

[0137] In the above <Example 1>, the kinase was overexpressed in the inducible resistance monoclonal cells and treated with osimertinib (OSI), a third-generation EGFR targeting agent, to confirm cells in which resistance was induced.

[0138] Specifically, the same method as in <Example 2> was performed except that osimertinib 1 μM was treated as an EGFR targeting agent.

[0139] As a result, as shown in Fig. 3 and Table 1, 23 kinases and their genes were identified among 589 kinases that were repeatedly expressed in PC9 cells in which osimertinib resistance was induced. In particular, 16 kinases were confirmed to be commonly expressed in erlotinib and osimertinib resistance.

[0140] Kinases commonly involved in inducing resistance to erlotinib and osimertinib: FGR, SYK, CSF1R, FES, CRKL, AXL, YES1, FRK, RAF1, FGFR2, ARAF, ALK, PDGFRA, ITK, PIK3CG, HCK

[0141] The above results demonstrate that the 16 kinases are involved in inducing resistance to erlotinib and osimertinib. Therefore, the cell line overexpressing these kinases was used as a cell model system for screening drugs that suppress resistance to EGFR-targeted anticancer agents.

[0142] <Example 4> Production of a re-resistant cell model after withdrawal from EGFR-targeted therapy

[0143] <4-1> Acquisition of drug-responsive cells after EGFR-targeted therapy withdrawal

[0144] In the above <Example 2>, kinase-overexpressing PC9-1 cells in which erlotinib resistance was induced were given a drug-free period against erlotinib, and then re-treated with erlotinib to confirm drug responsiveness.

[0145] Specifically, in the above <Example 2>, 1×10 kinase-overexpressing PC9-1 cells induced with erlotinib resistance were seeded in a 6-well plate. 5 After dispensing cells / well and adding 2 ml of culture medium (RPMI medium + 10% FBS + 1% penicillin-streptomycin antibiotics), the cells were subcultured at 3-day intervals with erlotinib removed, and a resting period was provided. After 6 months of subculture, the cells were seeded in a 96-well plate at a density of 5 × 10 3Cells were seeded at 10 cells / well and cultured for 24 h. Then, erlotinib was treated at concentrations of 0, 0.001, 0.003, 0.01, 0.033, 0.1, 0.33, 1, and 3.3 μM for 120 h. After 120 h, the MTT assay was performed using an MTT assay kit (Invitrogen) according to the manufacturer's procedure to confirm drug reactivity.

[0146] As a result, as shown in Fig. 4, it was confirmed that drug responsiveness to erlotinib was restored in cells overexpressing 10 types of kinases.

[0147] <4-2> Creation of a re-resistant cell model after EGFR-targeted therapy withdrawal

[0148] In the above Example <4-1>, 10 types of kinase overexpressing cells that recovered drug responsiveness were treated with erlotinib for 7-24 days, and then drug responsiveness was confirmed.

[0149] Specifically, in the above Example <4-1>, 10 kinds of kinase overexpressing cells showing erlotinib sensitivity were seeded at 1×10 in a 6-well plate. 5 Cells were seeded at 10 cells / well and cultured for 24 hours with 2 ml of culture medium (RPMI medium + 10% FBS + 1% Penicillin-Streptomycin). After 24 hours, the culture medium was replaced with a new one and 1 μM of erlotinib was treated. Thereafter, the culture medium and erlotinib were replaced with new ones at 3-day intervals. After 14 days, the cells with induced resistance were transferred to a 10 cm cell culture plate and cultured for 7 days with 1 μM of erlotinib to induce complete erlotinib resistance. Then, to confirm whether complete resistance to erlotinib was actually induced, the cells were seeded in a 96-well plate at a density of 5 × 10 3Cells were dispensed per well and drug responsiveness was confirmed using the same method as described in Example <4-1> above. As a control group, 10 kinase-overexpressing cells (cell lines that had not been exposed to erlotinib after overexpression of 10 genes) were used.

[0150] As a result, as shown in Fig. 5, it was confirmed that drug resistance appeared by re-treatment with erlotinib in 10 types of kinase-overexpressing cells that recovered drug responsiveness, and it was confirmed that re-resistance appeared more quickly compared to the control group.

[0151] The above results demonstrate that the 10 kinases are involved in the induction of resistance to erlotinib after a drug withdrawal period. Therefore, the cell line overexpressing these kinases and restoring drug responsiveness was used as a cell model system for screening drugs that suppress resistance to EGFR-targeted anticancer agents.

[0152] <Example 5> Identification of resistance regulation mechanisms in an EGFR-targeted therapy-resistant cell model.

[0153] After performing RNA-profiling using a cell model that overexpressed the 10 kinases identified in the above Example <4-2> and recovered drug responsiveness, GSEA (https: / www.gsea-msigdb.org / gsea / index.jsp) provided by the Broad Institute was performed to analyze the signaling pathway transmission system regulated according to drug responsiveness.

[0154] As a result, as shown in Fig. 6, the MYC target pathway was commonly identified to show an increase (resistance), decrease (drug holiday period), and increase (re-resistance) pattern depending on the drug response to erlotinib in cell models overexpressing ALK, AXL, and FES, and MYC and ODC1 were identified as factors that mainly regulate the pathway (leading edge genes).

[0155] In addition, after performing RNA-profiling using a cell model in which the 6 kinases (CRKL, SYK, AXL, ERBB2, FES, ALK) of the above <Example 2> were overexpressed and erlotinib resistance was induced, GSEA (https: / www.gsea-msigdb.org / gsea / index.jsp) provided by the Broad Institute was performed to analyze the signaling pathway transmission system regulated according to drug responsiveness.

[0156] As a result, as shown in Fig. 7, it was confirmed that the polyamine pathway was commonly increased compared to the control group, and ODC1 was identified as a key gene regulating the polyamine pathway.

[0157] Accordingly, using the pooled shRNA screening technique capable of targeting 5,000 genes, a pooled shRNA virus was produced in a cell model that overexpressed ALK and recovered drug response as confirmed in Example <4-2> above, and after calculating the efficiency, it was introduced according to a method commonly used in the art so that one virus could enter one cell at a virus efficiency that could reach MOI = 0.3. Thereafter, 1 μM of erlotinib was treated, and re-resistance was induced using the same method as described in Example <4-2> above, and then genes that were reduced or disappeared among the 5,000 genes were analyzed.

[0158] As a result, as shown in Figure 8, the condition with reduced ODC1 expression was ranked highest, confirming that re-resistance to erlotinib did not occur. The condition with reduced MYC expression was also confirmed to not result in re-resistance to erlotinib.

[0159] In addition, the 5 kinases (AXL, ALK, CSF1R, FES, YES1) confirmed in the above Example <4-2> were overexpressed, and cells that recovered drug response were transferred to a 10 cm cell culture plate and cultured in a condition containing 1 μM of erlotinib to induce erlotinib re-resistance. In addition, cells were harvested at 4-day intervals from day 1 to day 20 of culture, and after protein extraction from the cells using a method conventional in the art, Western blotting was performed to confirm the expression of MYC and ODC1 proteins.

[0160] As a result, as shown in Fig. 9, it was confirmed that the expression of MYC and ODC1 increased as resistance was acquired.

[0161] Through the above results, it was confirmed that the polyamine pathway involving MYC and OCD1 is important when resistance to the EGFR targeting therapy is acquired by overexpression of the kinase involved in inducing resistance to the anticancer drug, and in particular, ODC1 involved in the polyamine pathway plays an important role.

[0162] <Example 6> Confirmation of inhibition of acquisition of resistance to first-generation EGFR targeted therapy by inhibition of polyamine pathway regulators.

[0163] In the above <Example 5>, it was confirmed that the polyamine pathway involving ODC1 and MYC is important when resistance to the treatment is acquired by overexpression of kinases involved in inducing resistance to the anticancer agent EGFR targeted therapy. Accordingly, ODC1 was selected as a priority as a candidate target for suppressing resistance to EGFR targeted therapy, and the suppression effect of acquisition of resistance to first-generation EGFR targeted therapy by ODC1 suppression was confirmed, and the suppression effect of acquisition of resistance to first-generation EGFR targeted therapy by MYC suppression was also additionally confirmed.

[0164] <6-1> Confirmation of inhibition of acquisition of resistance to first-generation EGFR targeted therapy by ODC1 inhibition

[0165] A cell model was created in which the expression of ODC1, identified in <Example 5> above as a candidate target for suppressing resistance to EGFR-targeted therapeutics as an anticancer agent, was suppressed, and after treating the model with erlotinib, suppression of acquisition of drug resistance was confirmed.

[0166] Specifically, a tet-on system vector capable of regulating the expression of ODC1 shRNA by doxycycline was constructed. Using the constructed vector, a virus was constructed in the same manner as described in <Example 2>, and the constructed virus was infected into the FGFR1 or ERBB2 overexpressing cells of <Example 2> as a cell model for screening drugs that inhibit EGFR targeted therapy resistance. Then, hygromycin selection (500 ㎍ / ml) was performed for 7 days to select only virus-infected cells. The virus-infected cells are ODC1-shRNA knockdown cells in which ODC1 shRNA is expressed by doxycycline treatment and ODC1 expression is suppressed. The selected cells were seeded in a 96-well plate at a density of 5 × 10 3 Cells were seeded at 1 μM / well and treated with 1 μM erlotinib. After 48 h, the culture medium was replaced with fresh erlotinib, and 2 μg / ml doxycycline (- / + condition) was treated to suppress ODC1 expression together with 1 μM erlotinib. Afterwards, cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining at 2-day intervals.

[0167] As a result, as shown in Fig. 10, the number of cells in the erlotinib and doxycycline combination treatment group (ER+shODC1+dox) was reduced by more than 50% compared to the erlotinib alone treatment group (ER), confirming that acquisition of resistance to erlotinib was suppressed.

[0168] <6-2> Confirmation of inhibition of re-acquisition of resistance after withdrawal of first-generation EGFR targeted therapy by MYC inhibition

[0169] A cell model of drug response recovery during the first-generation EGFR targeted therapy withdrawal period was created by suppressing the expression of MYC, which was identified in <Example 5> above as a target for suppressing resistance to EGFR targeted therapy as an anticancer agent, and suppressing acquisition of drug resistance was confirmed after treating the model with erlotinib.

[0170] Specifically, a tet-on system vector capable of regulating the expression of MYC shRNA by doxycycline was constructed, and a virus was constructed using the same method as described in Example <6-1>. Thereafter, the constructed virus was infected with 10 types of kinase-overexpressing cells that had recovered drug responsiveness in Example <4-1>, using the same method as described in Example <6-1>.

[0171] As a result, as shown in Figure 11, the number of cells in all 10 cell types was reduced in the erlotinib and doxycycline combination treatment group (ER+shMYC) compared to the erlotinib single treatment group (ER), confirming that acquisition of resistance to erlotinib was suppressed.

[0172] Through the results of the above <Example 6>, it can be seen that the polyamine pathway is important when resistance to the first-generation EGFR targeted therapeutic agent is acquired by overexpression of a kinase involved in inducing resistance to the anticancer agent, and that acquisition of resistance to the first-generation EGFR targeted therapeutic agent is suppressed by suppression of the pathway-involving factor, ODC1 or MYC, particularly by suppression of ODC1.

[0173] <Example 7> Confirmation of restoration of responsiveness to first-generation EGFR targeted therapy by inhibition of polyamine pathway regulators.

[0174] As in the above <Example 6>, ODC1 was selected as a priority as a candidate target for suppressing resistance to EGFR targeted therapy as an anticancer agent, and the effect of restoring the responsiveness of the first-generation EGFR targeted therapy by suppressing ODC1 was confirmed, and the effect of restoring the responsiveness of the first-generation EGFR targeted therapy by suppressing MYC was additionally confirmed.

[0175] <7-1> Confirmation of restoration of responsiveness to first-generation EGFR targeted therapy following ODC1 inhibition

[0176] As a candidate target for suppressing resistance to EGFR targeted therapy as an anticancer agent, an erlotinib-resistant cell model was created by suppressing the expression of ODC1, identified in <Example 5> above, and drug responsiveness was confirmed after treating the model with erlotinib.

[0177] Specifically, the virus produced in the above Example <6-1> was infected into a cell model in which erlotinib resistance was induced and overexpressed the 29 genes of the above Example 2. Next, virus-infected cells were selected using the method described in the above Example <6-1>, and the selected cells were treated with erlotinib, and then 48 hours later, erlotinib and doxycycline (- / + conditions) were treated. Thereafter, the number of cells was measured at two-day intervals.

[0178] As a result, as shown in Figure 12, among the cells in which erlotinib resistance was induced overexpressing 29 kinases (CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK, BTK), 27 (CSF1R, RAF1, SRC, ALK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK, BTK), excluding 2 types (ITK, PIK3CG), Resistance to erlotinib was overcome in cells overexpressing kinases (FGFR2, YES1, MOS, NTRK3, MST1R, CRKL, FRK, BLK, BTK), resulting in erlotinib responsiveness.

[0179] <7-2> Confirmation of recovery of responsiveness to first-generation EGFR targeted therapy following MYC inhibition

[0180] To suppress resistance to EGFR targeted therapy as an anticancer agent, an erlotinib-resistant cell model was created by suppressing the expression of MYC, which was identified in <Example 5> above, and drug responsiveness was confirmed after treating the model with erlotinib.

[0181] Specifically, the virus produced in the above Example <6-2> was infected into 11 cell models in which erlotinib resistance was induced and overexpressed the 29 genes of the above Example 2. Then, the experiment was performed using the same method as described in the above Example <7-1>. In addition, in order to determine the correlation between MYC and ODC1 in the polyamine pathway, cells were harvested after the experiment, and protein was extracted from the cells using a method commonly used in the art, and Western blotting was performed to confirm the expression of MYC and ODC1 proteins.

[0182] As a result, as shown in Fig. 13, among the cells in which erlotinib resistance was induced by overexpressing 11 kinases (CSF1R, RAF1, SRC, ALK, ARAF, FGR, FGFR2, YES1, PIK3CG, FRK, CRKL), resistance to erlotinib was overcome in cells overexpressing 10 kinases, excluding cells overexpressing CRKL, and erlotinib responsiveness was exhibited.

[0183] Additionally, as shown in Figure 14, ODC1 protein expression was reduced by suppression of MYC expression in cells with erlotinib resistance that overexpress seven kinases (CSF1R, ALK, ARAF, FGR, FGFR2, FRK, CRKL).

[0184] The results of <Example 7> above demonstrate that the polyamine pathway is crucial when resistance to first-generation EGFR-targeted anticancer drugs is acquired through the overexpression of kinases involved in the induction of resistance to the aforementioned therapeutic agents. Furthermore, MYC and ODC1, which are involved in the pathway, are crucial, and ODC1 is regulated by MYC, and thus, inhibition of ODC1 restores the responsiveness of first-generation EGFR-targeted drugs.

[0185] <Example 8> Confirmation of restoration of responsiveness to third-generation EGFR targeted therapy by inhibition of polyamine pathway regulators.

[0186] As in the above <Example 6>, ODC1 was selected as a priority as a candidate target for suppressing resistance to EGFR targeted therapy as an anticancer agent, and the effect of restoring responsiveness to the third-generation EGFR targeted therapy by suppressing ODC1 was confirmed, and the effect of restoring responsiveness to the third-generation EGFR targeted therapy by suppressing MYC was additionally confirmed.

[0187] <8-1> Confirmation of restoration of responsiveness to third-generation EGFR targeted therapy following ODC1 inhibition

[0188] As a candidate target for suppressing resistance to EGFR targeted therapy as an anticancer agent, an osimertinib-resistant cell model was created by suppressing the expression of ODC1, identified in <Example 5> above, and drug responsiveness was confirmed after treating the model with osimertinib.

[0189] Specifically, the virus produced in Example <6-1> was infected into a cell model in which osimertinib resistance was induced and overexpressed the 16 genes of Example 3. Then, the experiment was performed in the same manner as described in Example <7-1>, except that osimertinib was used instead of erlotinib.

[0190] As a result, as shown in Fig. 15, among the cells in which osimertinib resistance was induced by overexpressing 16 kinases (HCK, RAF1, SYK, FGFR2, ITK, PIK3CG, CSF1R, FGR, ARAF, ALK, CRKL, YES1, PDGFRA, AXL, FES, FRK), resistance to osimertinib was overcome in cells overexpressing 13 kinases (CSF1R, ALK, ITK, AXL, PDGFRA, ARAF, FGR, FES, FGFR2, YES1, PIK3CG, CRKL, FRK), excluding cells overexpressing HCK, RAF1, or SYK, and osimertinib responsiveness was exhibited.

[0191] <8-2> Confirmation of recovery of responsiveness to third-generation EGFR targeted therapy following MYC inhibition

[0192] To suppress resistance to EGFR targeted therapy as an anticancer agent, an osimertinib-resistant cell model was created by suppressing the expression of MYC, as identified in <Example 5> above, and drug responsiveness was confirmed after treating the model with osimertinib.

[0193] Specifically, the experiment was performed in the same manner as described in Example <8-1>, except that the virus produced in Example <6-2> was infected with the virus. In addition, in order to investigate the correlation between MYC and ODC1 in the polyamine pathway, cells were harvested after the experiment, and protein was extracted from the cells using a method commonly used in the art, and Western blotting was performed to confirm the expression of MYC and ODC1 proteins.

[0194] As a result, as shown in Fig. 16, resistance to osimertinib was overcome in cells overexpressing 13 types of kinases, excluding cells overexpressing HCK, RAF1 or SYK, similar to the above Example <8-1>, and osimertinib responsiveness was exhibited.

[0195] Additionally, as shown in Figure 17, ODC1 protein expression was reduced by suppression of MYC expression in cells with osimertinib resistance that overexpress 16 types of kinases.

[0196] The results of <Example 8> above demonstrate that the polyamine pathway is crucial when resistance to third-generation EGFR-targeted anticancer agents is acquired through the overexpression of kinases involved in the induction of resistance to the agent. Furthermore, MYC and ODC1, which are involved in the pathway, are crucial, and ODC1 is regulated by MYC, and thus, inhibition of ODC1 restores the responsiveness of third-generation EGFR-targeted agents.

[0197] <Example 9> Increased apoptosis markers confirmed in a model of EGFR-targeted therapeutic response recovery following ODC1 inhibition.

[0198] Polyamines are known to regulate programmed cell death (PN Moschou et al. Journal of Experimental Botany, Vol. 65, No. 5, pp. 1285-1296, 2014). Therefore, we confirmed cell death in a model of EGFR-targeted therapeutic response restoration through inhibition of ODC1, which is involved in the polyamine pathway.

[0199] <9-1> Increased apoptosis markers confirmed in a model of recovery of responsiveness to first-generation EGFR targeted therapy following ODC1 inhibition.

[0200] In the above Example <7-1>, the expression of apoptosis markers was confirmed in a cell model in which responsiveness to erlotinib was restored by ODC1 inhibition.

[0201] Specifically, after performing the same method as described in Example <7-1> above, protein was extracted from cells using a method commonly used in the art, and then Western blotting was performed.

[0202] As a result, as shown in Fig. 18, it was confirmed that cleaved PARP, an apoptosis marker, increased by doxycycline treatment in 16 cells (NTRK2, MAP2K1, SYK, HCK, MST1R, NTRK3, FGFR1, NTRK1, ERBB2, PRKCQ, FGR, BTK, YES1, ALK, RAF1, BLK) among the cells in which erlotinib resistance was induced overexpressing 21 kinases.

[0203] <9-2> Increased apoptosis markers confirmed in a model of recovery of responsiveness to third-generation EGFR targeted therapy following ODC1 inhibition.

[0204] In the above Example <8-1>, the expression of apoptosis markers was confirmed in a cell model in which drug responsiveness to osimertinib was restored by ODC1 inhibition.

[0205] Specifically, after performing the same method as described in the above Example <8-1>, protein was extracted from cells using a method commonly used in the art, and then Western blotting was performed.

[0206] As a result, as shown in Fig. 19, it was confirmed that cleaved PARP increased by doxycycline treatment in 13 types of cells (FRK, AXL, ALK, PIK3CG, YES1, SYK, CSF1R, FGR, FGFR2, FES, ARAF, RAF1, PDGFRA) among the cells in which erlotinib resistance was induced overexpressing 16 types of kinases.

[0207] Through the results of the above <Example 9>, it can be seen that the polyamine pathway is important when resistance to the anticancer drug is acquired by overexpression of a kinase involved in inducing resistance to the EGFR targeted therapy, and that the recovery of the responsiveness to the EGFR targeted therapy is due to cell death caused by regulation of the polyamine pathway by the reduction of ODC1 and regulation of ODC1 by MYC, a factor involved in the pathway.

[0208] <Example 10> Confirmation of inhibition of acquisition of resistance to first-generation EGFR targeted therapy by treatment with an ODC1 inhibitor in a cell model.

[0209] As in <Example 6> above, after treating DFMO with an ODC1 inhibitor using cells overexpressing FGFR1 or ERBB2, inhibition of acquisition of erlotinib resistance was confirmed.

[0210] Specifically, the FGFR1 or ERBB2 overexpressing cells of the above <Example 2> were seeded at 5×10 in a 96-well plate. 3 Cells were seeded at 10 cells / well and co-treated with 1 μM erlotinib and 25 μM or 50 μM DFMO 48 hours later. Cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining at 2-day intervals.

[0211] As a result, as shown in Figure 20, it was confirmed that the number of cells was reduced in the erlotinib and DFMO combination treatment group (ER+DFMO) compared to the erlotinib alone treatment group (ER), thereby suppressing the acquisition of resistance to erlotinib.

[0212] <Example 11> Confirmation of restoration of EGFR-targeted therapeutic response by ODC1 inhibitor treatment in a cell model

[0213] <11-1> Confirmation of restoration of responsiveness to first-generation EGFR targeted therapy by ODC1 inhibitor treatment in a cell model

[0214] Using a cell model in which erlotinib resistance was induced by overexpressing the kinase of the above <Example 2>, drug responsiveness to erlotinib was confirmed after co-treatment with DFMO and erlotinib as an ODC1 inhibitor.

[0215] Specifically, 5×10 cells with erlotinib resistance that overexpress the 28 genes of the above <Example 2> were seeded in a 96-well plate. 3 Cells were seeded at 1 cell / well and co-treated with 1 μM erlotinib and 50 μM DFMO 48 hours later. Afterwards, cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining at 2-day intervals.

[0216] As a result, as shown in Fig. 21, it was confirmed that the responsiveness to erlotinib was restored through a decrease in the number of cells induced to be resistant to erlotinib by combined treatment with erlotinib and DFMO in cells overexpressing 28 types of kinases.

[0217] <11-2> Confirmation of restoration of responsiveness to third-generation EGFR targeted therapy by ODC1 inhibitor treatment in a cell model

[0218] Using a cell model in which osimertinib resistance was induced by overexpressing the kinase of the above <Example 3>, drug responsiveness to osimertinib was confirmed after co-treatment with DFMO and osimertinib as an ODC1 inhibitor.

[0219] Specifically, cells with osimertinib resistance induced by overexpressing the 16 genes of <Example 3> above were seeded in a 96-well plate at a density of 5×10 3 Cells were seeded at 1 cell / well and co-treated with 1 μM osimertinib and 50 μM DFMO 48 hours later. Afterwards, cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining at 2-day intervals.

[0220] As a result, as shown in Figure 22, it was confirmed that the responsiveness to osimertinib was restored through a decrease in the number of cells induced to be resistant to osimertinib by combined treatment with osimertinib and DFMO in cells overexpressing 15 types of kinases except for one type (PDGFRA).

[0221] In addition, using a cell model in which osimertinib resistance was induced by overexpressing the kinase of the above <Example 3>, drug responsiveness to lazertinib was confirmed after co-treatment with DFMO as an ODC1 inhibitor and the third-generation EGFR targeting drug lazertinib.

[0222] First, the drug responsiveness of DFMO and lazertinib was confirmed in PC9 cells, a non-small cell lung cancer cell line. Specifically, 1 × 10 PC9 cells were seeded in a 96-well plate. 3Cells were seeded at 10 cells / well and treated with vehicle, 1 μM lazertinib, or 10 μM or 15 μM DFMO 24 h later. After 2-day intervals, cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining, and the results were graphed as a percentage of cell growth compared to the vehicle-treated group.

[0223] In addition, among the cells in which osimertinib resistance was induced by overexpressing the 23 genes of the above <Example 3>, 5 types of cells were seeded in a 96-well plate at 5×10 3 Cells were seeded at 10 cells / well and treated 24 hours later with 1 μM lazertinib alone or in combination with DFMO (10 μM or 15 μM). Afterwards, cell counts were measured using Cytation5 after DAPI (Hoechst 33342, Invitrogen) staining at 2-day intervals, and the cell growth percentage was plotted compared to the lazertinib-only group.

[0224] As a result, as shown in Figure 23, in PC9 cells in which osimertinib resistance was not induced, drug response to lazertinib was observed and cell growth was inhibited when lazertinib was treated (Figure 23, left). In contrast, osimertinib-resistant cells treated with lazertinib alone did not show drug response to lazertinib, and osimertinib-resistant cells treated with a combination of lazertinib and DFMO showed restored drug response to lazertinib (Figure 23, right).

[0225] <Example 12> Confirmation of inhibition of acquisition of resistance to first-generation EGFR targeted therapy by administration of ODC1 inhibitor in animal model

[0226] Inhibition of drug resistance acquisition was confirmed after co-administration of DFMO with ODC1 inhibitor in a mouse model transplanted with FGFR1 or ERBB2 overexpressing cells.

[0227] Specifically, 5×10 FGFR1 or ERBB2 overexpressing cells of <Example 2> were each injected into 5-week-old Balb / c nude mice. 6 Xenograft mice were created by subcutaneously transplanting cells. Control mice were transplanted with PC9 cells. Eight mice were grouped for each condition, and it took one week for tumors to form. The average tumor size was 100 mm. 3 When the target was reached, the control mice (PC9) were administered vehicle (30% PEG solution), erlotinib (25 mg / kg), or DFMO (1%), and the FGFR1-overexpressing cell xenograft mouse group (FGFR1) and the ERBB2-overexpressing cell xenograft mouse group (ERBB2) were administered vehicle, erlotinib, or erlotinib and DFMO. The vehicle and erlotinib were administered orally daily, and DFMO was supplied in the drinking water.

[0228] As a result, as shown in Figure 24, the PC9 group showed no effect of single administration on DFMO, and the FGFR1 and ERBB2 groups showed tumor growth from 30 days after single administration of erlotinib, and completely acquired resistance at 55 days. On the other hand, it was confirmed that the FGFR1 and ERBB2 groups did not completely develop resistance under conditions of combined administration of erlotinib and DFMO.

[0229] <Example 13> Confirmation of restoration of responsiveness to first- and third-generation targeted therapeutics by administration of ODC1 inhibitors in animal models.

[0230] To determine whether the administration of an ODC1 inhibitor overcomes resistance to EGFR-targeted anticancer drugs and restores drug response, we co-administered DFMO as an ODC1 inhibitor to mouse models transplanted with erlotinib-resistant cells overexpressing FGFR1 or ERBB2 and mouse models transplanted with osimertinib-resistant cells overexpressing YES1 or AXL, and confirmed tumor growth inhibition.

[0231] Specifically, 5 × 10 cells overexpressing FGFR1 or ERBB2 of the above <Example 2> and 5 × 10 cells overexpressing YES1 or AXL of the above <Example 3> were each injected into 5-week-old Balb / c nude mice to induce erlotinib resistance. 6 Xenograft mice were created by subcutaneously transplanting cells. Five to nine mice were grouped for each condition, and tumors were formed one week later. The average tumor size was 100 mm. 3 When reached, the control mice (PC9) were administered vehicle (30% PEG solution), erlotinib (25 mg / kg), or DFMO (1%), and the FGFR1-overexpressing erlotinib-resistant induced cell xenograft mouse group (FGFR1) and the ERBB2-overexpressing erlotinib-resistant induced cell xenograft mouse group (ERBB2) were administered erlotinib, or erlotinib and DFMO. The vehicle and erlotinib were administered orally daily, and DFMO was supplied in drinking water. The YES1-overexpressing osimertinib-resistant induced cell xenograft mouse group (YES1) and the AXL-overexpressing osimertinib-resistant induced cell xenograft mouse group (AXL) were administered osimertinib (5 mg / kg) or osimertinib and DFMO.

[0232] As a result, as shown in Figure 25, it was confirmed that the FGFR1 group and the ERBB2 group showed continuous tumor growth when erlotinib was administered alone, whereas tumor growth was suppressed under conditions of combined administration of erlotinib and DFMO.

[0233] In addition, as shown in Figure 26, it was confirmed that the YES1 group and the AXL group showed continuous tumor growth when ocimone tip was administered alone, whereas tumor growth was suppressed under conditions of combined administration of ocimone tip and DFMO.

[0234] The above results indicate that resistance to first- or third-generation EGFR-targeted anticancer drugs can be overcome through ODC1 inhibition, thereby restoring drug responsiveness to these drugs.

[0235] <Example 14> Confirmation of increased ODC1 expression in EGFR-targeted therapy-resistant mouse tumor tissues and EGFR-targeted therapy-resistant patient tissues.

[0236] The expression level of ODC1 in mouse tumor tissues that acquired resistance to the first-generation EGFR targeted therapy erlotinib was confirmed through immunohistochemistry (IHC).

[0237] Specifically, the tumor tissue (Vehicle) of the FGFR1 or ERBB2 overexpressing cell xenograft mouse model obtained in the above <Example 12> under vehicle treatment conditions and the tumor tissue (ER) that acquired resistance through long-term administration of erlotinib of the FGFR1 or ERBB2 overexpressing cell xenograft mouse model were used to confirm the expression of ODC1 by performing IHC using a method commonly used in the art.

[0238] In addition, to investigate whether this phenomenon also occurs in patients, IHC was performed using a method commonly used in the art using tumor tissues of patients before and after acquiring erlotinib resistance obtained from Samsung Seoul Hospital, as shown in [Table 2] below, to confirm the expression of ODC1.

[0239] Pathology slide number: Before / after acquisition of EGFR-targeted therapy resistance S1204831 Before S1726971 Before S1628922 Before S1302723 Before S1603256 Before S1125883 Before S1444109 After Elotin S1423960 After Zephini Tip S1639568 After Elotin S1437126 After Zephini Tip S1423960 After Zephini Tip S1212940 After Zephini Tip

[0240] As a result, as shown in Figure 27, ODC1 was not expressed in the Vehicle group, but ODC1 expression was observed in the ER group. In addition, as shown in Figure 28, it was confirmed that ODC1 expression was increased in patients after acquiring resistance.

[0241] Through the results of the above <Examples 1> to <Examples 14>, it was confirmed that the polyamine pathway is important when resistance to the therapeutic agent is acquired by overexpression of a kinase involved in inducing resistance to the EGFR targeted therapy in cancer, specifically non-small cell lung cancer, and that MYC and ODC1 involved in the pathway play an important role in particular, and that ODC1 is regulated by MYC in the pathway. Accordingly, by regulating the pathway by suppressing ODC1 expression or activity, it was confirmed that acquisition of resistance to the EGFR targeted therapy agent due to overexpression of the kinase is suppressed, and resistance is alleviated and drug responsiveness is restored. Therefore, the ODC1 inhibitor that suppresses ODC1 expression or activity can be used as an active ingredient of a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

[0242] In the present invention, when EGFR targeted therapy resistance is induced using a non-small cell lung cancer cell model overexpressing a kinase involved in inducing EGFR targeted therapy resistance and an animal model xenografted with the same, it was confirmed that when ODC1 expression or activity is suppressed, acquisition of EGFR targeted therapy resistance due to kinase overexpression is suppressed, and resistance is alleviated, resulting in the effect of restoring drug responsiveness. Therefore, the agent that suppresses ODC1 expression or activity can be usefully used as an active ingredient of a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

Claims

1. A pharmaceutical composition containing an ODC1 inhibitor as an active ingredient for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

2. A pharmaceutical composition according to claim 1, wherein the ODC1 inhibitor inhibits ODC1 expression or activity.

3. A pharmaceutical composition according to claim 2, wherein the ODC1 inhibitor that suppresses ODC1 expression is selected from the group consisting of antisense nucleotides, small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) that complementarily bind to the mRNA of the ODC1 gene.

4. A pharmaceutical composition according to claim 2, wherein the ODC1 inhibitor that inhibits ODC1 activity is selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, and natural products that specifically bind to the ODC1 protein.

5. A pharmaceutical composition according to claim 4, wherein the compound that specifically binds to the ODC1 protein is DFMO.

6. A pharmaceutical composition according to claim 1, wherein the ODC1 inhibitor further suppresses or alleviates anticancer drug resistance after an anticancer drug withdrawal period.

7. A pharmaceutical composition according to claim 1, wherein the anticancer agent is an EGFR (Epidermal Growth Factor Receptor) targeted therapy agent.

8. A pharmaceutical composition according to claim 7, wherein the EGFR targeted therapeutic agent is at least one selected from the group consisting of erlotinib, gefitinib, afatinib, dacomitinib, osimertinib, lazertinib, pelitinib, neratinib, icotinib, brigatinib, lapatinib, canertinib, vandetanib, PKI-166, and AEE788.

9. A pharmaceutical composition according to claim 1, wherein the anticancer agent exhibits resistance in at least one cancer selected from the group consisting of breast cancer, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, squamous cell carcinoma, thyroid cancer, stomach cancer, bladder cancer, and glioblastoma.

10. A pharmaceutical composition according to claim 9, wherein the anticancer agent exhibits resistance in cancer having a high level of expression of one or more genes or proteins among CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK, and BTK compared to a normal control group.

11. A pharmaceutical composition for preventing or treating cancer resistant to anticancer drugs, containing an ODC1 inhibitor as an active ingredient.

12. A pharmaceutical composition according to claim 11, wherein the pharmaceutical composition further contains an anticancer agent.

13. An anticancer adjuvant for the prevention or treatment of anticancer drug-resistant cancer, containing an ODC1 inhibitor as an active ingredient. 14.1) A step of measuring the expression level of one or more genes or proteins thereof from a sample isolated from a cancer patient, including CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK; and 2) A method for providing information for predicting response and prognosis to an anticancer drug, comprising a step of judging that anticancer drug resistance will be suppressed by co-administering an anticancer drug and an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample. 15.1) A step of measuring the expression level of one or more genes or proteins thereof from a sample isolated from a patient with anticancer drug-resistant cancer, including CSF1R, RAF1, SRC, ALK, ITK, AXL, PRKCQ, NTRK1, PDGFRA, PDGFRB, NTRK2, HCK, ARAF, FGFR1, ERBB2, FGR, SYK, MAP2K1, FES, FGFR2, YES1, MOS, NTRK3, MST1R, PIK3CG, CRKL, FRK, BLK and BTK; 2) A method for providing information for predicting response and prognosis to an anticancer drug, comprising a step of judging that anticancer drug sensitivity will be restored by co-administering an anticancer drug and an ODC1 inhibitor when the expression level of the gene or its protein in step 1) is higher than that of the corresponding gene or its protein in a normal control sample.

16. Use of an ODC1 inhibitor for preparing a pharmaceutical composition for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity.

17. Use of an ODC1 inhibitor for preparing a pharmaceutical composition for preventing or treating anticancer drug-resistant cancer.

18. Use of an ODC1 inhibitor for preparing an anticancer adjuvant for the prevention or treatment of anticancer drug-resistant cancer.

19. A method for suppressing anticancer drug resistance, restoring anticancer drug responsiveness, or enhancing anticancer drug sensitivity, comprising administering an ODC1 inhibitor to a subject.

20. A method for preventing or treating cancer resistant to anticancer drugs, comprising administering an ODC1 inhibitor to a subject.

21. A method for preventing or treating cancer resistant to anticancer drugs, comprising administering an ODC1 inhibitor and an anticancer drug to a subject.

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