WO2019103238A1 - Agent thérapeutique contre le cancer résistant aux inhibiteurs de pi3k/mtor - Google Patents

Agent thérapeutique contre le cancer résistant aux inhibiteurs de pi3k/mtor Download PDF

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WO2019103238A1
WO2019103238A1 PCT/KR2017/015785 KR2017015785W WO2019103238A1 WO 2019103238 A1 WO2019103238 A1 WO 2019103238A1 KR 2017015785 W KR2017015785 W KR 2017015785W WO 2019103238 A1 WO2019103238 A1 WO 2019103238A1
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pi3k
mtor
bet
cells
inhibitor
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조광현
이호성
이수범
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한국과학기술원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to a novel cancer therapeutic agent, and more particularly, to a PI3K / mTOR inhibitor resistant cancer therapeutic agent and a screening method thereof.
  • Cancer is a representative invasive and intractable disease that has become a socioeconomic problem.
  • colorectal cancer CRC
  • CRC colorectal cancer
  • the rate of increase in the incidence of colorectal cancer due to Westernized eating habits and obesity has increased over the past 10 years Respectively.
  • the incidence and mortality rates of colorectal cancer patients are expected to increase more steeply.
  • the treatment of patients with metastatic colorectal cancer still fails to overcome the limitations of low patient response.
  • the PI3K / mTOR pathway is a signaling pathway that plays an important role in regulating cell proliferation, growth, and survival.
  • PI3K, mTOR, and KRAS which are major constituents of this signal transduction pathway, may cause cancer if abnormally increased function or hyperactivity occurs.
  • about 30% of patients with PI3K / mTOR Variations of the proteins located in the signal transduction pathway are observed.
  • PI3K / mTOR inhibitors have been developed for the purpose of inhibiting the proliferation of cancer and are in clinical trials, but their therapeutic effect is inferior due to the drug resistance which is necessarily caused by drug treatment.
  • Korean Patent Laid-Open Publication No. 2009-0090460 discloses an anticancer drug for colon cancer and an adjuvant for radiotherapy chemotherapy which contain imidazoquinoline.
  • the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a more effective treatment agent for resistant cancer against PI3K / mTOR inhibitor and a screening method for the therapeutic agent.
  • these problems are exemplary and do not limit the scope of the present invention.
  • a pharmaceutical composition comprising i) a BET inhibitor and a PI3K / mTOR inhibitor; Or ii) a PI3K / mTOR inhibitor comprising as an active ingredient a pharmaceutical composition for the treatment of a resistant cancer against a PI3K / mTOR inhibitor, wherein the BET inhibitor is covalently bound to the PI3K / mTOR inhibitor as an active ingredient. Or a pharmaceutically acceptable salt thereof.
  • a pharmaceutical composition comprising a pharmaceutically effective amount of i) a BET inhibitor and a PI3K / mTOR inhibitor; Or ii) administering the drug conjugate wherein the BET inhibitor is covalently bound to the PI3K / mTOR inhibitor to a patient suffering from an EGFR inhibitor resistant cancer.
  • a process for preparing a compound or natural product comprising: reacting a test compound or a natural substance with BET; And selecting a test compound or a natural substance that specifically binds to BET.
  • the present invention also provides a method of screening a candidate compound for resistance cancer treatment for a PI3K / mTOR inhibitor.
  • a method for treating BET comprising: treating a cell expressing BET with a test compound or a natural product; And a step of screening a test compound or natural product significantly inhibiting the expression of BET as compared to a control group that has not undergone any treatment, and a method for screening a candidate for a resistance cancer therapeutic agent for a PI3K / mTOR inhibitor.
  • a method for detecting a BET comprising: treating a test compound or a natural product with a composition comprising BET and a pair protein that interacts with the BET; And selecting a test compound or a natural substance that significantly inhibits the interaction of the BET and the parent protein as compared to a control group that has not been treated with any one of the methods for screening a candidate compound for a resistance cancer treatment candidate for a PI3K / mTOR inhibitor do.
  • the treatment efficiency of resistant intractable cancer against the PI3K / mTOR inhibitor can be drastically increased.
  • the scope of the present invention is not limited by these effects.
  • FIG. 1 is a graph showing the inhibition effect of double PI3K / mTOR on the cell viability of colorectal cancer cells.
  • the upper graph is a graph of GI50 values of various colorectal cancer (CRC) cells for BEZ235 treatment, (microsatellite instability) - instability, CpG methylation factor phenotype (CIMP) positive.
  • CRC colorectal cancer
  • FIG. 2A is an analysis of the inhibitory effect of double PI3K / mTOR on the cellular responses of colorectal cancer cells, and is a growth analysis graph for various CRC cells treated with the indicated concentrations of BEZ235 measured using IncuCyte.
  • Figure 2B is a graph of cell cycle analysis on various CRC cells treated with BEZ235 (500 nM) for 2 or 10 days using PI staining and flow cytometry. Untreated cells were used as controls. The data represent the mean ⁇ S.E.M. of the 3 biologic replicates. The three biological replicons p values were calculated between untreated cells and BEZ235 treated cells. *, p ⁇ 0.05; **, p ⁇ 0.01; Student t-test.
  • Figure 2c is an AUC graph of apoptotic curves normalized to the AUC of cell proliferation curves of cells treated with BEZ235 (500 nM) for 2 or 10 days in various CRC cells measured using IncuCyte.
  • Figure 2d is a graph of apoptotic cells treated with BEZ235 (500 nM) for 2 or 10 days in various CRC cells determined using Caspase-3/7 activity assay. Untreated cells were used as controls.
  • FIG. 3A is a graph showing that the dual PI3K / mTOR inhibition induces a dynamic adaptive kinome response in colorectal cancer cells.
  • a heat map (FIG. 3A) analyzing the array of phosphoric acid antibodies against HCT116 cells treated with BEZ235 (500 nM) for 2 or 10 days )to be.
  • FIG. 3B is a graph showing that the dual PI3K / mTOR inhibition induces a dynamic adaptive kinome response in colorectal cancer cells.
  • a heat map (FIG. 3B) analyzing the array of phosphoric acid antibodies on SW480 cells treated with BEZ235 (500 nM) for 2 or 10 days )to be.
  • Experimental medication samples were standardized for each DMSO-treated control.
  • Protein kinases with significant differences (p ⁇ 0.05, Student's t-test) between BEZ235- and DMSO-treated cells on days 2 or 10 are presented. Protein kinases were classified according to the Standard Kinase Classification Scheme.
  • Figure 3c shows Western blot analysis of the indicated signaling kinase of PI3K / mTOR pathway in HCT116 cells treated with various concentrations of BEZ235 for 1 hour.
  • Figure 3d shows Western blot analysis of the indicated signal kinase of PI3K / mTOR pathway in SW480 cells treated with BEZ235. AKT1 and GAPDH were used as loading controls.
  • Figure 3e is the Western blot results for some up-regulated tumor proteins from the phospho-antibody sequence analysis in HCT116 cells treated with BEZ235 (500 nM) for the indicated time (0-240 hr).
  • Figure 3f is the Western blot results for some up-regulated tumor proteins from the phospho-antibody sequence analysis in SW480 cells treated with BEZ235 (500 nM) for the indicated time (0-240 hr). GAPDH was used as a loading control.
  • Figure 4a shows that double PI3K / mTOR inhibition activates a variety of carcinogenic pathways and proteins in colorectal cancer cells, resulting in a mRNA level of the indicated gene in cells treated with BEZ235 at indicated doses (1-500 nM) for 24 hours
  • the results are shown in Fig.
  • the results were normalized to GAPDH level.
  • Data represent the mean of three biologic repeats + S.E.M. *, p ⁇ 0.05; **, p ⁇ 0.01; Student's t-test.
  • Figure 4b is the Western blot results for Myc of HCT116 or SW480 cells treated with BEZ235 (500 nM) for 2 or 10 days. GAPDH was used as a loading control.
  • FIG. 5A shows that double PI3K / mTOR inhibition induces cell senescence in colorectal cancer cells.
  • HCT116 and SW480 cells were treated with BEZ235 (500 nM) for 2, 7, or 10 days and then stained with? -Galactosidase It is a photograph.
  • FIG. 5B is a graph showing an average + S.E.M quantified from the three times biological replications of FIG. 5A.
  • FIG. p value was determined in comparison with control (day 0) cells.
  • FIG. 5c shows that the inhibition of double PI3K / mTOR induces cell senescence in colorectal cancer cells, indicating the percentage of ⁇ H2AX-positive cells treated with BEZ235 (500 nM) for 2, 7 or 10 days determined using FACS analysis Graph.
  • FIG. 5d shows that dual PI3K / mTOR inhibition leads to cell senescence in colorectal cancer cells, Western blot for phosphorylated p38, Chk1 and Chk2, and total of cells treated for 2 or 10 days with BEZ235 (500 nM) p16 and p21. GAPDH was used as loading control.
  • Figure 5e shows that dual PI3K / mTOR inhibition induces cell senescence in colorectal cancer cells and various CRC cells were stained with BEZ235 (1 ⁇ M, MEK inhibitor) or BIRB796 (1 ⁇ M, pan-p38 inhibitor) (500 nM) for 10 days and then stained with? -Galactosidase.
  • FIG. 5F is a graph showing an average + S.E.M quantified from the three times biological replicates of FIG. 5E.
  • FIG. 5F is a graph showing an average + S.E.M quantified from the three times biological replicates of FIG. 5E.
  • FIG. 5g shows that double PI3K / mTOR inhibition induces cell senescence in colorectal cancer cells.
  • HCT116 and SW480 cells were treated with GDC-0941 (1 ⁇ M, pan-PI3K inhibitor) or everolimus (1 ⁇ M, mTOR inhibitor) And then stained with? -Galactosidase.
  • FIG. 5H is a graph showing the mean + S.E.M quantified from the three times biological replicates of FIG. 5G.
  • Figure 5i shows that dual PI3K / mTOR inhibition leads to cell senescence in colorectal cancer cells, and it was confirmed that ⁇ H2AX positive cells treated for 10 days with BEZ235 GDC-0941 (1 ⁇ M) or everolimus (1 ⁇ M) determined using FACS analysis ≪ / RTI >
  • Figure 6a shows that dual PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomes.
  • FIG. 6B is a graph quantifying the average spot pixel density of the greater amount of the most up-regulated cytokines obtained in FIG. 6A using ImageJ software.
  • FIG. 6B is a graph quantifying the average spot pixel density of the greater amount of the most up-regulated cytokines obtained in FIG. 6A using ImageJ software.
  • FIG. 6C shows that double PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomes and cells treated with BEZ235 (500 nM) for 2 or 10 days And the protein secretion of the cytokine shown in FIG.
  • FIG. 6d shows that double PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomes.
  • FIG. 3 is a graph showing mRNA expression of the cytokine displayed.
  • Levels of protein secretion and mRNA expression were measured by ELISA and quantitative RT-PCR, respectively, and p-values were determined as compared to control (day 0) cells and data represent mean + S.E.M.
  • Figure 6e shows that double PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomas, indicating the absence or presence of U0126 (1 ⁇ M) and BIRB796 (1 ⁇ M) (500 nM) under the same conditions as in Example 1.
  • FIG. 6e shows that double PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomas, indicating the absence or presence of U0126 (1 ⁇ M) and BIRB796 (1 ⁇ M) (500 nM) under the same conditions as in Example 1.
  • FIG. 6f shows that double PI3K / mTOR inhibition-induced aged colorectal cancer cells develop ERK- and p38-regulated secretomes, and the absence or presence of U0126 (1 ⁇ M) and BIRB796 (1 ⁇ M)
  • FIG. 2 is a graph showing mRNA expression of cytokines indicated in cells treated with BEZ235 (500 nM). The levels of the protein secretion or mRNA expression were measured by ELISA and quantitative RT-PCR, respectively. p value was determined as compared to control BEZ235 treated cells.
  • FIG. 7a is a graph showing the effect of age-related secre- tomes on the drug sensitivity to PI3K / mTOR double inhibition in colorectal cancer cells.
  • the mean fluorescence was measured and the cells treated with BEZ235 (500 nM) (MFI) using a graphical FACS analysis of the expression levels of the cognate receptors on 8 (i. E., CXCR1 and CXCR2) and MIF (i. E., CD44 and CD74).
  • Figure 7b is an analysis of the effect of aging-related secre- tomes on drug susceptibility to PI3K / mTOR dual inhibition in colorectal cancer cells, with recombinant cytokines (100 ng / ml IL-8 and MIF) ≪ / RTI > is the Western blot analysis results for the indicated protein of stimulated cells over time.
  • Figure 7c is an analysis of the effect of senescence-related secre- tomes on drug susceptibility to PI3K / mTOR dual inhibition in colorectal cancer cells, showing the presence of BEZ235 (500 ⁇ g / ml) in the absence or presence of CXCR1 / 2 blocking antibody nM), AUC of the apoptosis curve normalized to the AUC of cell proliferation curve for normalization of cell number or ISO-1 (50 [mu] M, MIF inhibitor) was administered for 7 days. The cells were treated for 7 days with the indicated combination of drugs and the cell proliferation from the phase-contrast image using IncuCyte and the confluence of red fluorescence (number of apoptotic cells measured by PI) were measured.
  • Figure 7d shows the effect of aging-related secre- tomes on drug susceptibility to PI3K / mTOR dual inhibition in colorectal cancer cells using CXCR1 / 2 blocking antibody (2.5 ⁇ g / ml) or ISO-1 (50 ⁇ M) 3/7 activity of apoptotic cells treated with BEZ235 (500 nM) for 7 days in the presence or absence of the cells. Untreated cells were used as controls.
  • FIG. 7E is an analysis of the effect of aging-related secretion on drug sensitivity to PI3K / mTOR double inhibition in colorectal cancer cells.
  • BEZ235 treatment change in normalized magnification in the control group
  • GI50 value 8 or MIF secretion level with ERK1 / 2 activity normalized magnification change for control
  • p38 activity control-normalized multiple-change
  • Figure 7f shows the effect of age-related secre- tomes on drug susceptibility to PI3K / mTOR dual inhibition in colorectal cancer cells.
  • a CRC cell line with a GI50 value of greater or less than 400 nM BEZ235 treatment for 7 days
  • the mean level of secretion of the indicated cytokine In a CRC cell line with a GI50 value of greater or less than 400 nM, BEZ235 treatment for 7 days And the mean level of secretion of the indicated cytokine.
  • Figure 8a is a Venn diagram depicting the gene expression profiling analysis of the functional role of the senescence-related secretome induced by PI3K / mTOR block in colorectal cancer cells and the overlapping of DEGs of HCT116 and SW480 cells .
  • the p value of the intersection size of DEG was calculated using hypergeometric tests.
  • FIG. 8b is a graphical representation of the gene expression profiling in which the functional role of the aging-related secretome induced by PI3K / mTOR blocking in colorectal cancer cells is analyzed.
  • the gene ontology that is significantly enriched between DEGs upregulated in HCT116 cells A graph for the term GO term.
  • FIG. 8c is a graphical representation of the gene expression profiling in which the functional role of the aging-related secretome induced by PI3K / mTOR block in colorectal cancer cells is analyzed.
  • the gene ontology that is significantly enriched between DEGs upregulated in SW480 cells It is a graph for the term.
  • Figure 8d is a graphical representation of the gene expression profiling that analyzes the functional role of the aging-related secretome induced by PI3K / mTOR blockade in colon cancer cells.
  • GSEA Gene set enrichment analysis
  • FIG. 8E is a graph showing GSEA enrichment plots of gene sets displayed on SW480 cells in relation to gene expression profiling that analyzed the functional role of aging-related secretomers induced by PI3K / mTOR blockade in colorectal cancer cells.
  • Figure 9a shows that PI3K / mTOR inhibition-induced senescence shows tumorigenic activity in colorectal cancer cells. It shows that the CRC cells treated with DMSO, CM-DMSO or CM-BEZ in the absence or presence of BEZ235 (500 nM) Proliferation analysis graph.
  • FIG. 9B is a graph showing that PI3K / mTOR inhibition-induced senescence shows tumorigenic activity in colorectal cancer cells.
  • PI propidium iodide
  • FIG. 9c is a graph showing analysis of wound healing of cells treated with DMSO, CM-DMSO, or CM-BEZ for a specified period of time, in which PI3K / mTOR inhibition-induced senescence shows tumorigenic activity in colorectal cancer cells.
  • Figure 9d is a graph showing the mean + S.E.M quantified from the 3x biopile replicates of Figure 9c.
  • FIG. 9E is a graph showing the in vitro migration and infiltration of cells treated with DMSO, CM-DMSO or CM-BEZ for 48 hours in which PI3K / mTOR inhibition-induced senescence shows tumorigenic activity in colorectal cancer cells to be.
  • Figure 9f is a graph showing the mean + S.E.M quantified from the triplicate biological replicates of Figure 9e.
  • Figure 10A is a graph showing the proliferation of CRC cells treated with the indicated combination of drugs in which inhibition of PI3K / mTOR and BET combination induced apoptosis and analyzed cell sensitivity to treatment of PI3K / mTOR inhibitor.
  • Cell proliferation was measured over time by calculating cell confluence from multiphase-contrast images using IncuCyte.
  • FIG. 10B shows a graph showing the effect of combination inhibition of PI3K / mTOR and BET on cell number normalization in cells treated for 10 days with the indicated combination of drugs that induced apoptosis and analyzed cell sensitivity to treatment of PI3K / mTOR inhibitors
  • Cells were treated with the indicated drug combination for 10 days and the confluence of cell proliferation and red fluorescence (number of apoptotic cells measured by PI) from the phase-contrast image was measured using IncuCyte.
  • Figure 10d shows the results of a cell co-treatment with BEZ235 (500 nM) and JQ1 (1 [mu] M, BET inhibitor) in which the combined inhibition of PI3K / mTOR and BET induced apoptosis and assayed for cell sensitivity to treatment of PI3K / mTOR inhibitor Western blot (for 1 or 48 hours) of tumor proteins including multiple RTKs and signaling kinases. GAPDH was used as a loading control.
  • Figure 10e shows the inhibition of the combination of PI3K / mTOR and BET inducing apoptosis and analyzing the sensitivity of the cells to the treatment of PI3K / mTOR inhibitors.
  • the results showed that the inhibition of PI3K / mTOR inhibitors was inhibited by 1 day for RTK and cytokines in the absence or presence of JQ1 Lt; / RTI > is a graph showing mRNA expression levels of the indicated RTKs or cytokines in cells treated with BEZ235 (500 nM) for 7 days. Levels of mRNA expression were measured by quantitative RT-PCR.
  • Figure 10f shows the inhibition of the combination of PI3K / mTOR and BET inducing apoptosis and analyzing the sensitivity of the cells to the treatment of PI3K / mTOR inhibitors.
  • FIG. 11A is a graph showing the analysis of the gene track showing the occupation of BRD4 and H3K27ac in the indicated gene of the cell line indicated by analysis that BET inhibition blocked recruitment of BRD4 to the regulatory region of the multiple resistance mediator gene.
  • Sites A and B represent sites where primers were designed to test the binding of BRD4 in a ChIP-qPCR experiment.
  • FIG. 11B shows that BET inhibition blocks the recruitment of BRD4 to the regulatory region of the multi-resistance mediator gene, and HCT116 (upper layer) treated with BEZ235 (500 nM), JQ1 (1 [mu] M) And the ChIP-qPCR analysis of BRD4 binding at the indicated sites in SW480 (lower layer) cells.
  • FIG. 11D is a graph showing the analysis of the gene track showing the occupation of BRD4 and H3K27ac in the indicated gene of the cell line indicated by the analysis that BET inhibition blocked the recruitment of BRD4 to the regulatory region of the multiple resistance mediator gene.
  • Sites A and B represent sites where primers were designed to test the binding of BRD4 in a ChIP-qPCR experiment.
  • FIG. 11E shows that BET inhibition blocks the recruitment of BRD4 to the regulatory region of the multiple resistance mediator gene.
  • FIG. 12A is a graph showing the average weight change of mice treated with the combination of drugs indicated by analysis of antitumor efficiency of combination inhibition of PI3K / mTOR and BET in vivo.
  • FIG. 12A is a graph showing the average weight change of mice treated with the combination of drugs indicated by analysis of antitumor efficiency of combination inhibition of PI3K / mTOR and BET in vivo.
  • FIG. 12B is a graph showing the tumor growth curves of HCT116 and SW480 allografts treated with BEZ235, JQ1, or a combination thereof, in which the antitumor efficiency of inhibition of combination of PI3K / mTOR and BET in vivo is analyzed. *, p ⁇ 0.05, **, p ⁇ 0.01; One - way ANOVA.
  • FIG. 12C is a graph showing the average body weight of tumors excised from mice treated with BEZ235, JQ1, or a combination thereof, in which anti-tumor efficacy of combination inhibition of PI3K / mTOR and BET in vivo is analyzed. **, p ⁇ 0.01; Student's t-test.
  • PI3K / mTOR is a signaling pathway that plays a key role in regulating cellular responses such as cell survival, growth, migration, and metabolism, When controlled, cancer is induced.
  • BET protein inhibitor As used herein, the term " BET protein inhibitor" is widely used in studies in the US and Europe as one type of drug with anticancer, immunosuppressive and other effects in clinical trials. The inhibitor reversibly binds to bromodomain and the bromodomain of the BRD2, BRD3, BRD4, and BRDT proteins to prevent protein-protein interactions between BET protein and acetylated histones and transcription factors .
  • the term "functional fragment of an antibody” refers to a fragment of an antibody in which an antigen-binding site is conserved, not a full-length antibody as a heterodimer consisting of two heavy chains and two light chains,
  • the fragments include Fab, F (ab ') 2 , Fab', a single chain fragment variable (scFv), or a single-domain antibody (sdAb) derived from the antibody.
  • Fab is an antigen-binding antibody fragment that is produced by digesting an antibody molecule into a protease, papain, and is a dimer of two peptides of VH-CH1 and VL- , And other fragments generated by papain are referred to as Fc (fragment crystalisable).
  • F (ab ') 2 refers to a fragment comprising an antigen binding site in a fragment produced by digesting an antibody with pepsin, a protease, and the form of a tetramer in which two Fabs are linked by a disulfide bond .
  • Another fragment produced by pepsin is referred to as pFc '.
  • Fab &quot is produced by reducing the F (ab ') 2 as an antibody fragment having a locus similar to that of the Fab, and the length of the heavy chain portion is slightly longer than that of Fab.
  • scFv refers to a single chain fragment variable, a recombinant antibody fragment in which the variable regions (VH and VL) of the Fab of the antibody are made into a single strand using a linker.
  • sdAb single doamain antibody
  • sdAb single doamain antibody
  • a single variable region fragment of an antibody referred to as a nanobody.
  • the sdAb derived mainly from the heavy chain is used, but a single variable region fragment derived from the light chain has also been reported to be a specific binding to the antigen.
  • antibody mimetic is intended to include only heavy chains without light chains, unlike conventional full-length antibodies in which two heavy chains and two light chains form a quaternary structure of a hetero- Like protein produced from antibody fragments (VHH, VNAR, etc.) derived from camel or cartilaginous fish or protein scaffolds derived from non-antibodies such as nanobody, monobody, and variable lymphocyte receptor (VLR).
  • VHH antibody fragments
  • VNAR variable lymphocyte receptor
  • a pharmaceutical composition comprising i) a BET inhibitor and a PI3K / mTOR inhibitor; Or ii) a PI3K / mTOR inhibitor comprising a drug conjugate wherein said BET inhibitor is covalently bound to said PI3K / mTOR inhibitor as an active ingredient.
  • the BET-specific inhibitor may be a BET activity inhibitor or a BET expression inhibitor and the BET activity inhibitor may be a BET antagonist or an antibody functional fragment thereof, an antibody analogue specifically binding to BET, gallein or GRK2i.
  • the antibody functional fragment may be Fab, F (ab ') 2, Fab', scFv, Fv, sdAb, diabody or triabody and the antibody analogue may be selected from the group consisting of Affibody, Affilin, Affitin, Anticalin, DARPin, Fynomer, monobody, nanobody, VLR or VHH, and the BET expression inhibitor may be an antisense nucleotide, siRNA, shRNA or microRNA that specifically binds to the BET gene.
  • the resistant cancer for the PI3K / mTOR inhibitor may be a mutant or non-mutated cancer in the KRAS gene, and the mutation may be derived from exon 2 of the KRAS gene and the resistance to the PI3K / mTOR cancer Can be colon cancer.
  • the effective substance in the pharmaceutical composition for treating the PI3K / mTOR inhibitor resistant cancer, can be provided in the form of a single compound by covalently bonding the BEK inhibitor with the PI3K / mTOR inhibitor. Cases connected by covalent bonds while maintaining their functionality are well known in the art.
  • a drug conjugate in which an anticancer drug rapamycin is linked to a sigma-2 receptor-specific ligand, SV119, promotes apoptosis of cancer cells more than that of rapamycin alone (Spitzer et al ., Cancer Res ., 71 (1): 2012).
  • linkage between these small compounds occurs either directly through the amine group (-NH 2 ) and the carboxyl group (-COOH) amide bond contained in the compound, or through the formation of a polyethylene linker (- (CH 2 ) n -), a polyethylene oxide linker (CH 2 CH 2 O) n -).
  • the PI3K / mTOR inhibitor may be a PI3K / mTOR inhibitory compound, a PI3K / mTOR antagonistic antibody or an antibody functional fragment thereof, or an antibody analogue specifically binding to PI3K / mTOR, May be NVP-BEZ235, PI-103, PTEN, GSK3B, or HB9.
  • the antibody functional fragment may be Fab, F (ab ') 2 , Fab', scFv, Fv, sdAb, diabody or triabody and the antibody analogue may be selected from the group consisting of Affibody, Affilin, Affitin, Anticalin, DARPin, Fynomer, monobody, nanobody, VLR or VHH.
  • a pharmaceutical composition comprising a pharmaceutically effective amount of i) a BET inhibitor and a PI3K / mTOR inhibitor; Or ii) administering the drug conjugate wherein the BET inhibitor is covalently bound to the PI3K / mTOR inhibitor to a patient suffering from an EGFR inhibitor resistant cancer.
  • a process for preparing a compound or natural product comprising: reacting a test compound or a natural substance with BET; And selecting a test compound or a natural substance that specifically binds to BET.
  • the present invention also provides a method of screening a candidate compound for resistance cancer treatment for a PI3K / mTOR inhibitor.
  • a method for treating BET comprising: treating a cell expressing BET with a test compound or a natural product; And a step of screening a test compound or natural product significantly inhibiting the expression of BET as compared to a control group that has not undergone any treatment, and a method for screening a candidate for a resistance cancer therapeutic agent for a PI3K / mTOR inhibitor.
  • a method for detecting a BET comprising: treating a test compound or a natural product with a composition comprising BET and a pair protein that interacts with the BET; And selecting a test compound or a natural substance that significantly inhibits the interaction of the BET and the parent protein as compared to a control group that has not been treated with any one of the methods for screening a candidate compound for a resistance cancer treatment candidate for a PI3K / mTOR inhibitor do.
  • the mating protein may be selected from the group consisting of cyclin-dependent kinase 9 (CCK9), cyclin T1 (cyclin T1), cyclin T2 (cyclin T2), C15orf55 (protein NUT), hexamethylene bis- acetamide inducible 1 (lectin, galactoside-binding, soluble, 4), CDK2 (cyclin-dependent kinase 2), DNER (delta / notch-like EGF repeat), PLK1 (polo-like kinase 1), or LGALS4
  • the interaction between the BET and the paired protein can be determined by a yeast two hybrid assay, a fluorescence resonance energy transfer (FRET) assay, an affinity capture, a protein fragment complementary assay protein-fragment complementation assay (PCA), immunocoprecipitation (IP) analysis, or surface plasmon resonance assay.
  • CCK9 cyclin-dependent kinase 9
  • FRET fluorescence resonance energy transfer
  • PCA
  • a method of detecting BET comprising: measuring the degree of expression of BET from a tissue sample or a body fluid sample obtained from a cancer patient;
  • a method for predicting the PI3K / mTOR resistance of a cancer patient comprising predicting a cancer patient having an expression level of the BET higher than a normal level as having resistance to a PI3K / mTOR inhibitor.
  • a method of detecting BET comprising: measuring the degree of expression of BET from a tissue sample or a body fluid sample obtained from a cancer patient; Selecting cancer patients whose BET expression levels are higher than normal levels; And a step of simultaneously administering a PI3K / mTOR inhibitor and a BET inhibitor to a cancer patient selected as having a high expression level of BET.
  • a method of detecting a test compound comprising: reacting a test compound with BET and PI3K / mTOR, respectively; And screening a test compound that binds to both the BET and the active site of the PI3K / mTOR.
  • the present invention also provides a screening method for a candidate therapeutic substance for an EGFR inhibitor resistant cancer.
  • the test compound may be designed by a structure-based drug design.
  • the structure-based drug design uses a three-dimensional structure by computer modeling of BET and PI3K / mTOR to confirm whether a compound of a specific structure binds to the active site of BET and / or PI3K / mTOR, If not, the structure of the compound can be changed by the help of a computer.
  • a compound whose structure is determined by the help of a computer can be used to confirm whether the compound belongs to a known library when it is purchased and to have an actual activity. In the case of a novel compound, .
  • test compound inhibits its activity by binding to the actual BET can be confirmed by using the above-described various BET-small compound interaction assay method and / or BET-interacting pair protein interaction assay method have.
  • test compound inhibits the activity of PI3K / mTOR can be analyzed using a method similar to the method of analyzing whether the test compound inhibits the activity of BET.
  • PI3K / mTOR binds to a specific test compound is determined by radioimmunoassay labeling-affinity chromatography-affinity chromatography, drug affinity responsive target stability (DARTS) (DSRF), differential scanning fluorometry (DSF), or differential radial capillary action of ligands (SPROX), differential static light scattering ligand assay, DraCALA).
  • DARTS drug affinity responsive target stability
  • DSF differential scanning fluorometry
  • SPROX differential radial capillary action of ligands
  • DraCALA differential static light scattering ligand assay
  • the screening method comprising the steps of: treating the selected test compound with PI3K / mTOR resistant cancer cells overexpressing BET; And selecting a test compound that inhibits the growth of PI3K / mTOR resistant cancer cells.
  • a method for treating BET and PI3K / mTOR comprising: treating a test compound with cells expressing BET and PI3K / mTOR; Screening a candidate compound for a resistance to cancer treatment for a PI3K / mTOR inhibitor, comprising the step of screening a test compound that simultaneously inhibits the expression of GNB5 and PI3K / mTOR or inhibits its activity simultaneously as compared to a control not treated with any Method is provided.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the type of the affected part of the patient, the application site, the number of treatment, the treatment time, the formulation, the condition of the patient,
  • the amount to be used is not particularly limited, but may be 0.01 ⁇ g / kg / day to 10 mg / kg / day.
  • the above-mentioned daily dose may be administered once a day or two or three times a day at appropriate intervals, or intermittently administered at intervals of several days.
  • the pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluents which may be contained in an amount of 0.1 to 100% by weight based on the total weight of the composition and are conventionally used in the production of a pharmaceutical composition.
  • suitable carriers excipients and diluents which may be contained in an amount of 0.1 to 100% by weight based on the total weight of the composition and are conventionally used in the production of a pharmaceutical composition.
  • solid pharmaceutical preparations or liquid pharmaceutical preparations can be used for the preparation of pharmaceutical compositions.
  • the preparation additive may be either organic or inorganic.
  • excipients include lactose, sucrose, saccharose, glucose, cornstarch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide.
  • binder examples include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin and pectin, and the like.
  • lubricant examples include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals.
  • These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.
  • the pharmaceutical composition of the present invention can be prepared by any of the formulations conventionally produced in the art (for example, Remington's Pharmaceutical Science (latest edition; Mack Publishing Company, Easton PA), the form of the formulation is not particularly limited .
  • These formulations are generally known prescription Seo literature [Remington's Pharmaceutical Science, 15 th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 all Agency: is described in (Chapter 87 Blaug, Seymour).
  • the pharmaceutical composition of the present invention can be administered orally or parenterally.
  • parenteral administration is performed by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, Injection and intraperitoneal injection.
  • the inventors of the present invention found that when treating PI3K / mTOR inhibitor with colorectal cancer cells, the cancer-induced kinase proteins such as EGFR, IGF1R, FAK, and ERK and IL-8 and MIF The cytokine was activated and the drug resistance was confirmed.
  • the results of the simultaneous treatment of the PI3K / mTOR inhibitor and the BET inhibitor, which inhibits the activity of BET as a result of the simultaneous treatment of various types of colorectal cancer cells with various cancer-induced proteins and cytokines, And thus the resistance to the PI3K / mTOR inhibitor is effectively overcome such that the growth of cancer cells is strongly inhibited and the death is greatly increased.
  • BET inhibitors are more effective at overcoming resistance to PI3K / mTOR inhibitors than kinase inhibitors.
  • the BET inhibitor was shown to be a target protein that can effectively overcome resistance to PI3K / mTOR inhibitors regardless of mutation in colorectal cancer patients.
  • both the PI3K / mTOR inhibitor and the BET protein Inhibition of BET protein inhibitor effectively inhibited the growth of cancer cells and induces strong death, and furthermore, the secretion of cancer-promoting cytokines and growth factors was also effectively inhibited.
  • the efficacy of the drug combination was also confirmed in a tumor transplantation mouse model. Therefore, the inventors of the present invention discovered that the BET inhibitor can be applied to patients suffering from colon cancer caused by abnormality and activation of the PI3K / mTOR signaling pathway by the combined treatment with the PI3K / mTOR inhibitor.
  • Phosphoproteome analyzes were performed using Phospho Explorer Antibody Array (Full Moon BioSystems, Sunnyvale, Calif., USA) containing 1,318 protein antibodies according to the manufacturer's instructions. Briefly, cell lysates obtained from cells treated with DMSO or BEZ235 for the indicated days were harvested using protein extraction buffer (Full Moon BioSystems, Sunnyvale, Calif., USA) Full Moon BioSystems, Sunnyvale, Calif., USA). Antibody microarray slides were then treated with blocking solution (Full Moon BioSystems, Sunnyvale, CA, USA) for 30 min at room temperature and washed with Milli-Q grade water.
  • microarray slides were then incubated with biotinylated cell lysates in coupling solution (Full Moon BioSystems, Sunnyvale, Calif., USA) for 2 hours at room temperature. After coupling, the slides were washed six times with 30 ml of wash solution (Full Moon Biosystems, Inc.) and the biotinylated proteins bound using Cy3-conjugated streptavidin (GE Healthcare, Chalfont St.
  • RNA sequencing cells were treated with conditioned medium (CM) from aged or non-aged cells for 72 hours and total RNA of cells was isolated using Trizol reagent (Invitrogen, Carlsbad, Calif., USA) . RNA quality was assessed with an Agilent 2100 bioanalyzer using an RNA 6000 nanochip (Agilent Technologies, Amstelveen, The Netherlands) and RNA quantitation was performed using an ND-2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) .
  • the Bowtie 2 index was generated by representative transcription or genomic sequence analysis for genomic and transcriptome alignment.
  • An alignment file was used to collect transcripts, estimate their abundance, and detect different expression of genes.
  • Differentially expressed genes are based on the coefficients obtained from multiple alignments using the coverage of BedTools (Quinlan AR et al., Bioinformatics. 26 (6): 841-2.
  • the read number data was processed based on a quantile-quantile normalization method using edgeR (Robinson MD et al., Bioinformatics. 26 (1): 139-40. 2010).
  • Azelastine, vorinostat, pinometostat, UNC669, azathioprine, azithromycin, azithromycin, azithromycin, azithromycin, azithromycin, azithromycin, Propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and purchased from Selleckchem (Houston, TX, USA).
  • GSK2879553, lapatinib, OSI- Human IL-8 and MIF were purchased from Peprotech (Rocky Hill, NJ, USA).
  • ISO-1 was purchased from (Cambridge, MA, USA) Abcam phospho-EGFR (Tyr 869), EGFR, phospho-IGF1R (Tyr 1165/1166), IGF1R ⁇ , p16Ink4a, p21Cip1, Myc, phospho-4E-BP1 (Ser 65 ), rabbit IgG, and GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
  • Antibodies to CD74 and phospho-FAK (Tyr 910) is Abcam in (Cambridge, MA, USA), CD44, phospho-ERK (Thr 202 / Tyr 204), phospho-AKT1 (Thr 308), phospho-AKT1 (Ser 473 ), phospho-p70S6K (Thr 389 ), and phospho-p38 (Thr 180 / Tyr 182 ) were purchased from Cell Signaling Technology (Beverly, Mass., USA). Antibodies against BRD4 were purchased from Bethyl Laboratories (Montgomery, TX, USA).
  • mice IgG and human monoclonal anti-CXCR1 and anti-CXCR2 antibodies were detected in R & D Systems (Minneapolis, MN, USA), antibodies to phospho-Chk1 (Ser 317 ) and phospho-Chk2 (Thr 68 ) Invitrogen (Rockford, Ill., USA).
  • Human colorectal cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Va., VA, USA) except for the HCT116 and Caco-2 cell lines obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) Purchased and cultured according to the guidelines of ATCC and KCLB.
  • ATCC American Type Culture Collection
  • VA Manassas, Va., VA, USA
  • HCT116 and Caco-2 cell lines obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) Purchased and cultured according to the guidelines of ATCC and KCLB.
  • Cell viability assays were performed in 96-well plates and treated with 6 different concentrations of drug ranging from 0.1 nM to 1 [mu] M to determine the 50% inhibitory concentration (GI 50 ) of the indicated drug (BEZ235) The plate was incubated for 96 hours, and relative cell viability was measured.
  • the absorbance at 450 nm was measured using a xMarkMicroplate absorbance spectrophotometer (Bio-Rad, Hercules, CA, USA) after adding WST-1 solution (Daeillab, Seoul, Korea) Respectively. Each experiment was performed three times for each cell line and GI50 value was calculated using CalcuSyn (Biosoft, Cambridge, UK).
  • CI combination index
  • the cells were inoculated in triplicate in 96-well plates and treated with the indicated combination of drugs in the presence of PI.
  • the cells were then placed in a cell incubator at 37 ° C and 5% CO 2 and monitored using an IncuCyte live cell imaging system (Essen Bioscience, Ann Arbor, MN, USA).
  • the apparatus captures high quality phase contrast images of the cells and calculates a monolayer confluence of each image over a plurality of designated time points using a contrast-based merging algorithm.
  • Cell proliferation was measured over time by calculating cell confluence from multi-phase-contrast images acquired every three hours.
  • the curve for apoptotic cells was determined from the total percentage of red (relative to the number of apoptotic cells measured by PI) fluorescence.
  • the area of the curve for apoptotic cells (AUC) was normalized to the area of the cell proliferation curve for normalization of cell number using GraphPad Prism software (San Diego, Calif., USA).
  • Cells were treated with the indicated drug for the indicated period for cell cycle analysis. The cells were then trypsinized and washed twice with ice-cold phosphate-buffered saline (PBS) and fixed at -20 ° C with 70% ethanol overnight. After fixation, cells were resuspended in PBS containing RNAse a (100 ⁇ g / ml) and PI (50 ⁇ g / ml) and further incubated for 30 min at 37 ° C in the dark. Flow cytometry data were collected using CellQuest software (BD Biosciences, San Jose, Calif., USA) and analyzed using FlowJo software (Treestar, San Carlos, CA, USA).
  • PBS ice-cold phosphate-buffered saline
  • the detection of the cell ⁇ H2AX was carried out using a ⁇ H2AX phosphorylation assay kit (Millipore, Bedford, Mass., USA) using a FACSCalibur system (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.
  • CXCR1 and CXCR2 cells were incubated with anti-CXCR1 (MAB330, R & D Systems, Minneapolis, MN, USA), anti-CXCR2 (MAB331, R & D Systems, Minneapolis, (H + L) -Alexa Fluor 647 (Invitrogen, Carlsbad, Calif., USA) for 30 min.
  • CD44 and CD74 Cell Signaling Technology, Danvers, Mass., USA
  • anti-CD74 Abcam, Cambridge, MA, USA
  • mouse IgG antibody R & D Systems, Minneapolis, MN
  • Becton Dickinson, San Jose, Calif., USA Becton Dickinson, San Jose, Calif., USA
  • goat anti-mouse IgG H + L
  • Alexa Fluor 488 Invitrogen, Carlsbad, CA, USA
  • Caspase-3/7 activity was determined using Caspase-Glo 3/7 Assay Systems (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, the cells were plated in 96-well plates and treated for the indicated time with the indicated combination of drugs. Caspase-Glo 3/7 reagent was then added to each well and the cells were incubated at room temperature for 1 hour. The luminescence intensity of each well was then measured using an Orion II Microplate Luminometer (Berthold Detection System GMBH, Pforzheim, Germany).
  • RNA-spin RNA-spin according to the manufacturer's protocol (iNtRON Biotech, Seoul, Korea). Approximately 1 ⁇ g of RNA treated with DNAse I for 20 minutes was used to generate complementary DNA (cDNA) using a DiaStar RT kit (Solgent, Daejeon, Korea). The samples were then analyzed using a 20 mL reaction volume of StepOnePlus (Applied Biosystems, Waltham, MA, USA) containing cDNA, primers and SYBR Master Mix (Applied Biosystems, Waltham, Mass., USA) The experimental results were determined as the mean of three independent experiments and the data were normalized to GAPDH mRNA in each reaction. Sequence information of primers used for amplification of the quantitative real-time PCR and all the primers used in the present invention are summarized in Table 1 below.
  • the cells were treated with the indicated drug combination on plastic. The cells were then plated on 12-well plates at a density of 1 x 10 5 cells per well and stained using Senescence ⁇ -galactosidase staining kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Senescent cells were identified as blue-stained cells by light microscopy and were photographed with a phase contrast microscope in 10 randomly selected areas and the average ratio of ⁇ -galactosidase positive cells was determined .
  • Senescence ⁇ -galactosidase staining kit Cell Signaling Technology, Danvers, MA, USA
  • CM conditioned media
  • DMSO Dulbecco's Modified Eagle's Medium
  • the senescence-associated secretory phenotype which is metabolically active and collectively regulates the senescence-associated secretory phenotype (SASP), although the growth-inhibited aging cancer cells induced by therapy are inhibited under stressful conditions such as radiation or chemotherapy.
  • SASP senescence-associated secretory phenotype
  • PI3K / mTOR inhibition maintains the SASP characteristics in regulating the cellular microenvironment by over-secretion of cytokines and growth factors known as cytokines.
  • the expression profiles of the cytokines were analyzed in the supernatants of cells treated with BEZ235 for 10 days through array analysis.
  • cytokines in the supernatant of cells was performed using the Proteome Profiler Array Human XL Cytokine Array Kit (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. ImageJ software (Schneider et al., Nat Methods. 9 (7): 671-5, 2012) was used to quantify the average spot pixel density of cytokines.
  • IL-8 and MIF were measured using a human IL-8 / CXCL8 Quantikine ELISA kit (R & D Systems, Minneapolis, MN, USA) and a human MIF Quantikine ELISA Kit (R & D Systems, Minneapolis, Respectively.
  • ERK1 / 2 and phosphorylated levels of p38 are ERK1 / ERK2 (Total / Phospho) InstantOne TM ELISA kit (eBioscience, San Diego, CA, USA) and p38 MAPK [Total / Phospho (Thr 180 / Tyr 182)] InstantOne TM ELISA Kit (eBioscience, San Diego, Calif., USA) according to the manufacturer's instructions.
  • GSEA Gene Set Enrichment Analysis
  • GSEA analysis was performed to investigate the biological functions altered by SAS in colorectal cancer cells. For this, the gene was ranked by -log 10 (p value) multiplied by log 2 (magnification change) between the experimental and control samples. Thus, the most significantly up-regulated genes belonged to the higher class and the significantly down-regulated genes belong to the lower class.
  • GSEA software v3.0 (Subramanian A et al., Proc . Natl . Acad . Sci . USA. 102 (43): 15545-15550, 2005) was used to perform a pre-evaluated GSEA analysis and the number of permutations was 5,000 .
  • the set of genes used in the GSEA analysis was obtained from MSigDB (Liberzon A et al., Bioinformatics. 27 (12): 1739-1740, 2011).
  • Cells treated with BEZ235 (500 nM), JQ1 (1 ⁇ M) or a combination thereof for 8 hours were cross-linked with 1% formaldehyde for 10 min at room temperature and then incubated with 125 mM glycine glycine. The cells were then washed three times with PBS and the cells were lysed for 10 minutes in cold-lysis buffer (25 mM HEPES (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40 and protease inhibitor cocktail).
  • cold-lysis buffer 25 mM HEPES (pH 7.8), 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40 and protease inhibitor cocktail
  • the cells were harvested on ice using an ultrasonic buffer (50 mM HEPES (pH 7.8), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium dioxycholate, 0.1% SDS and protease inhibitor cocktail) , And ultrasonicated for 30 seconds using a sonicator (Diagenode, Denville, NJ, USA) for a total treatment time of 15 minutes and a 30 second off-cycle condition.
  • an ultrasonic buffer 50 mM HEPES (pH 7.8), 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% sodium dioxycholate, 0.1% SDS and protease inhibitor cocktail
  • the chromatin solution was preliminarily removed with 20 ⁇ l protein A + G magnetic beads (Milipore, Billerica, MA, USA) for 2 hours at 4 ° C and 20 ⁇ l protein A + G magnetic beads and 4 ⁇ g anti- Or anti-rabbit IgG was added and incubated overnight.
  • the beads were sequentially washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer and the bound materials were eluted with ChIP (Chromatin Immunoprecipitation) elution buffer (100 mM NaHCO3, 1% SDS) and incubated overnight at 65 ° C for cross-links.
  • ChIP Chromatin Immunoprecipitation
  • ChIP DNA was purified using MinElute PCR purification kit (Qiagen, Valencia, CA, USA). ChIP-qPCR was performed using SYBR green reagent and ABI 7500 Real-time PCR system (Applied Biosystems, Carlsbad, Calif., USA). To detect BRD4 binding to the site, primers for upstream of the TSS region of EGFR, IGF1R and MIF rich in BRD4 occupation were used for the ChIP-qPCR experiment (site A).
  • Cells were plated in 24-well transwell plates (8- ⁇ m pore size, Costar, Cambridge, Mass., USA) to measure cell mobility and invasiveness. 1x10 < 5 > cells were added to the upper chamber with a 0.1% gelatin coated membrane for migration assays. For invasion assays, chamber inserts were coated with Matrigel (BD Biosciences, San Jose, Calif., USA), dried overnight under aseptic conditions and 1 ⁇ 10 5 cells were added to the upper chamber. In these quantitative assays, the cells were suspended in serum-free medium and medium supplemented with indicated conditioned medium or DMEM containing 10% FBS was used as the chemoattractant in the lower chamber.
  • Matrigel BD Biosciences, San Jose, Calif., USA
  • non-mobile or non-invasive cells remaining on the upper surface of the membrane were removed by scraping after incubation for 48 hours at 37 ° C, 5% CO 2.
  • the cells on the lower surface of the membrane were fixed with 10% formaldehyde for 30 minutes and then stained with 0.1% crystal violet. Images were then taken with a phase contrast microscope in 10 randomly selected fields and the average number of cells migrated or invaded per field was quantified.
  • HCT116 and SW480 cells were suspended in a 1: 1 mixture of PBS and BD Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, Calif., USA) for CRT cell orthotopic engraftment and HCT116 and SW480 cells
  • Female nude mice Orient Bio Inc., Seongnam, Korea
  • mice with xenografts were treated with 7-8 mice per group when the average tumor volume reached 100 mm 3 (HCT116) and 250 mm 3 (SW480) Were selected randomly.
  • Example 1 duplex PI3K / mTOR Inhibition of cell proliferation in colorectal cancer cells
  • the most sensitive cell lines were Colo205, Colo201, HT29, and Caco2, and the most insensitive cell lines were HCT116, SW480, SW620, and LS180, while GI 50 of BEZ235 (50% inhibitory concentration of maximal cell proliferation) ranged from about 170 to 710 nM appear.
  • Double PI3K / mTOR inhibitory effect on cell viability of colorectal cancer cells condition Cell line GI 50 value [nM] KRAS BRAF PIK3CA APC CTNNB1 FBXW7 HCT116 712.70 mut wt mut wt mut wt SW480 645.26 mut wt wt mut wt wt SW620 589.08 mut wt wt mut wt wt LS180 543.63 mut mut mut wt mut wt LS174T 522.72 mut mut mut wt mut LOVO 505.88 mut wt wt mut wt wt Colo320-DM 484.33 wt wt wt mut wt wt HCC2998 428.07 mut wt wt mut wt mut SW1417 384.78 wt mut wt mut wt wt w
  • the present inventors tested BEZ235 at various concentrations (1 to 1,000 nM) with the four most insensitive (HCT116, SW480, SW620, and LS180) and the most sensitive 2 (HT29 and Caco2) CRC cell line and cell growth and survival were monitored using a live-cell imaging proliferation assay. As a result, it was observed that the treatment of BEZ235 inhibited cell growth under clear concentration-dependent conditions (Fig. 2A). In addition, although treatment with BEZ235 induced proper concentration of cells in the G1 phase of the cell cycle (Fig.
  • caspase-3 which is a reliable indicator of apoptosis in short-term (2 days) or even long- 7 activity or apoptosis did not increase (Fig. 2c and 2d).
  • the results show that treatment with BEZ235 exhibits cytoprotective effect rather than cytotoxic effect irrespective of the mutation state of CRC cells, so that CRC cells are resistant to apoptosis in response to PI3K / mTOR blocking, Suggesting that alternative regulatory mechanisms may exist to support survival.
  • Example 2 Double PI3K / mTOR Dynamic compensation in colorectal cancer cells by inhibition kinome Induction of reaction
  • Cancer cells activate compensatory survival-promoting mechanisms induced by activation of multiple protein kinases to avoid therapeutic effects of drug treatment.
  • HCT116 and SW480 cells were investigated the effect of two of the most insensitive CRC cell lines, HCT116 and SW480 cells on PI3K / mTOR blockade, to investigate the dynamic changes of kinase activity that contribute to the survival of CRC cells under PI3K / mTOR inhibition Signaling profiles were observed on the phosphoproteomic scale.
  • the kinase kinase of the cells responded significantly to BEZ235 treatment with many protein kinases that exhibited increased or decreased activity compared to untreated cells (Figs. 3a and 3b).
  • BEZ235 has been shown to induce multiple survival proteins, multiple RTKs (EGFR, insulin like growth factor 1 receptor, IGF1R), platelet-derived growth factor receptor (PDGFR) a ⁇ , vascular endothelial growth factor (VEGFR) Activation of cytoplasmic kinases (FAK, ERK, PAK (p21-active kinase) and Paxillin (PXN)).
  • RTKs EGFR, insulin like growth factor 1 receptor, IGF1R
  • PDGFR platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor
  • FAK cytoplasmic kinases
  • ERK ERK
  • PAK p21-active kinase
  • PXN Paxillin
  • HCT116 and SW480 CRC cells showed some variability in cellular response upon PI3K / mTOR blockade: PDGFR ⁇ / ⁇ , VEGFR2 and PAK were activated only in HCT116 cells whereas HER2, MET and PXN were activated in SW480 cells And 3b).
  • Example 3 Double PI3K / mTOR Induction of cellular senescence in colorectal cancer cells by inhibition
  • the present inventors have studied phenotypic behaviors of how cancer cells survive long-term PI3K / mTOR inhibition because they have inherent plasticity that can be adapted to adverse conditions such as chemotherapy and radiation therapy Respectively. For this, we observed the cell behavior of CRC cells under BEZ235 treatment and observed that BEZ235 treated cells exhibited flattened cell morphology, which is a typical feature of senescent cells. To investigate whether the cells actually become senescent, we observed whether ⁇ -galactosidase activity, which is a reliable marker of cell senescence, increases in cancer cells.
  • Example 4 duplex PI3K / mTOR Of aging colon cancer cells induced by inhibition ERK - and p38-regulated Seckley Tom (secretome) development
  • Treatment-induced aging cancer cells are cytokines, which are growth inhibited under stressful conditions such as radiation or chemotherapy, but remain metabolically active and collectively known as the age-related secretory phenotype (SASP) And regulates the cellular microenvironment by overexpressing growth factors.
  • SASP age-related secretory phenotype
  • ERK and p38 are known to act as upstream regulators of IL-8 and MIF expression at the transcriptional or translational level in a cellular-context dependent manner.
  • cytokines contribute to the resistance of CRC cells to PI3K / mTOR inhibition
  • the inventors first investigated whether the cells expressed cognate receptors of cytokines: IL-8 CXCR1 and CXCR2 for MIF, and CD44 and CD74 for MIF.
  • the expression levels of CXCR2 and CD74 were significantly increased after long term (10 days) BEZ235 treatment in both HCT116 and SW480 cells (Fig. 7A).
  • stimulation of cells with IL-8 or MIF activated several oncogenic downstream kinases induced in response to PI3K / mTOR blockade ( Figure 7b).
  • IL-8 and MIF were known to be functionally important in regulating survival and growth in a variety of cancer types
  • the inventors investigated the inhibitory effect of the cytokine on cellular responses of BEZ235-treated CRC cells.
  • inhibition of IL-8 or MIF activity using CXCR1 / 2 blocking antibody or ISO-1 (MIF antagonist) increased the induction of apoptosis in cells treated with BEZ235 (FIGS. 7C and 7D).
  • CRC cell lines with GI 50 values greater than 400 nM secreted significantly higher levels of cytokines compared to less than 400 nM (see Figure 7f, Tables 9 and 10). This indicates that the cytokine secretion profile is closely related to the drug susceptibility to PI3K / mTOR inhibition.
  • the activity of ERK1 / 2 activity was significantly higher in the cell line with a GI 50 value greater than 400 nM (see Tables 9 and 10), compared to less than 400 nM, indicating that ERK1 / 2 Suggesting that there is a positive correlation with activity.
  • CM-BEZ CM-induced cell
  • GSEA Gene set enrichment analysis
  • GSEA Gene set enrichment analysis
  • Example 6 PI3K / mTOR Inhibition of colorectal cancer cells by bulb- Tumor formation activation
  • TFs transcription factors
  • TFs transcription factors
  • the present inventors have decided to target epigenetic factors, proteins that modify or associate chromatin, in order to effectively prevent resistant cell responses at the transcription level and to control the expression of various genes.
  • various inhibitors of various epigenetic modulators have been tested and BET family bromodomains (Stratikopoulos EE et al., Cancer Cell. 27 ( 6): 837-51, 2015; Delmore JE et al., Cell 146 (6): 904-17, 2011) (see Tables 15 and 16).
  • BEZ235 and JQ1 significantly inhibited cell growth and increased apoptosis compared to drug alone (see Figures 10a and 10b and Tables 15 and 16).
  • BEZ235 Three drug treatments, BEZ235, OSI-906 (IGF1R inhibitor) and lapatinib (EGFR inhibitor), inhibited cell growth more effectively and increased apoptosis, but less than BEZ235 / JQ1 treatment, As well as the activation of additional genes or proteins that may contribute to resistance to PI3K / mTOR inhibition in CRC cells (Tables 17 and 18).
  • Combination treatment with BEZ235 and BIRB796 reduced BEZ235-induced growth inhibition but increased apoptosis, which could benefit from inhibition of TIS on CRC cells with increased proliferation, but at the same time due to reduction in global survival SAS Suggesting that they may lose their ability to survive.
  • BET inhibitors exhibit high specificity for BET family proteins (BRD2, BRD3, BRD4, and BRDT) compared to other bromodomain containing subfamilies and inhibit binding to acetylated lysine residues in histones.
  • BET family proteins BET2, BRD3, BRD4, and BRDT
  • chromatin immunoprecipitation sequencing ChIP-seq
  • RD4 and H3K27ac were enriched occupancy in RD4 and H3K27ac in the upstream regulatory region of the transcriptional start site (TSS) of EGFR, IGF1R and MIF in HCT116 and SW480 cells suggesting that this gene could be a direct target of BRD4 (Fig. 11A).
  • TSS transcriptional start site
  • regions rich in BRD4 and H3K27ac were not observed in the IL-8 gene (Fig. 11a). This indicates that IL-8 activity is indirectly regulated by BRD4 and BET inhibition inhibits IL-8 activation partially through ERK inhibition (Fig. 10C).
  • BET inhibition can regulate the expression of other resistance-mediated genes that are up-regulated by BEZ235 treatment in CRC cells, since BET inhibition is known to reduce the transcriptional activation of various genes (Fig. As a result, it was observed that JQ1 treatment inhibited BEZ235-induced upregulation of mRNA levels of CD44 and HES1 (Fig. 11C). In addition, the enriched occupancy of BRD4 and H3K27ac was also observed in HCT116 and SW480 cells in distinct regulatory regions upstream of the TSS of the gene (Region A), further suggesting the possibility of the gene as a direct target of BRD4 11d).
  • Example 8 In vivo ( in vivo ) PI3K / mTOR And BET
  • HCT116 and SW480 cells were subcutaneously injected to the back of nude mice and grown to an average size of 100 mm 3 (HCT116) and 250 mm 3 (SW480). The mice were then treated daily with 25 mg / kg / day of BEZ235 and / or 50 mg / kg / day of JQ1. As a result, no significant decrease in the mouse body weight was observed, suggesting that the experimental animals can withstand the treatment of the drug combination (FIG. 12A).
  • the pharmaceutical composition according to one embodiment of the present invention can be used for the manufacture of a medicament for the treatment of cancer having resistance to PI3K / mTOR inhibitors such as cancer, especially colon cancer.
  • the nucleic acid sequences shown in SEQ ID NOS: 1 to 38 are nucleic acid sequences of primers for RT-PCR for confirming the expression levels of various genes such as EGFR.

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Abstract

La présente invention concerne un nouvel agent thérapeutique contre le cancer et, plus particulièrement, un agent thérapeutique contre le cancer résistant aux inhibiteurs de PI3K/mTOR et un procédé de criblage associé.
PCT/KR2017/015785 2017-11-23 2017-12-29 Agent thérapeutique contre le cancer résistant aux inhibiteurs de pi3k/mtor WO2019103238A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140011222A1 (en) * 2009-02-10 2014-01-09 Nodality, Inc. Multiple mechanisms for modulation of the p13 kinase pathway
US20140080810A1 (en) * 2010-11-15 2014-03-20 Exelixis, Inc. Benzoxazepines as Inhibitors of PI3K/mTOR and Methods of Their Use and Manufacture
WO2015070020A2 (fr) * 2013-11-08 2015-05-14 Dana-Farber Cancer Institute, Inc. Polythérapie pour le traitement du cancer utilisant des inhibiteurs de protéine à bromodomaine et à domaine extra-terminal (bet)

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140011222A1 (en) * 2009-02-10 2014-01-09 Nodality, Inc. Multiple mechanisms for modulation of the p13 kinase pathway
US20140080810A1 (en) * 2010-11-15 2014-03-20 Exelixis, Inc. Benzoxazepines as Inhibitors of PI3K/mTOR and Methods of Their Use and Manufacture
WO2015070020A2 (fr) * 2013-11-08 2015-05-14 Dana-Farber Cancer Institute, Inc. Polythérapie pour le traitement du cancer utilisant des inhibiteurs de protéine à bromodomaine et à domaine extra-terminal (bet)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDREWS, FOREST H. ET AL.: "Dual-activity PI3K-BRD4 Inhibitor for the Orthogonal Inhibition of MYC to Block Tumor Growth and Metastasis", PNAS, vol. 114, no. 7, 30 January 2017 (2017-01-30), pages E1072 - E1080, XP055485319 *
STRATIKOPOULOS, ELIAS E. ET AL.: "Kinase and BET Inhibitors Together Clamp Inhibition of PI3K Signaling and Overcome Resistance to Therapy", CANCER CELL, vol. 27, no. 6, 2015, pages 837 - 851, XP029166214, doi:10.1016/j.ccell.2015.05.006 *

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