KR20190051744A - An anticancer agent for treating cancers resistant to EGFR inhibitor - Google Patents

Abstract

The present invention relates to an EGFR inhibitor-resistant cancer therapeutic agent, capable of remarkably increasing the treatment efficiency of resistant refractory cancers and, more specifically, to a pharmaceutical composition for treating a resistant cancer against an EGFR inhibitor, which comprises a GNB5 inhibitor and an EGFR inhibitor, or a drug liner as an active ingredient.

Description

An anticancer agent for treating cancers resistant to EGFR inhibitor

The present invention relates to a novel cancer therapeutic agent, and more particularly, to a therapeutic agent effective for resistance cancer against an EGFR inhibitor and a screening method thereof.

Colorectal cancer (CRC) is one of the most common types of cancer in the world. The overall 5-year relative survival rate of colorectal cancer patients in the localized stage is almost 90%. However, when progress is initiated, the 5-year survival rate is known to decrease to about 20% (Siegel et al ., Cancer J. Clin ., 62 (4): 220-241, 2012). The prognosis of patients with metastatic colorectal cancer (mCRC) remains poor despite therapeutic advances in the median overall survival time of 18-21 months (Bardelli and Siena, J. Clin. Oncol. 28 (7): 1254-1261, 2010). This has led to much effort to identify more effective drug targets for mCRC.

Cetuximab (trade name Erbitux  ) Is a monoclonal antibody to the epithelial growth factor receptor (EGFR) and is used to treat CRC patients by inhibiting EGFR. The EGFR signaling pathway plays an important role in various cellular functions such as cell growth, differentiation, survival, cell cycle progression and angiogenesis. KRAS is a signaling molecule downstream of the EGFR pathway, and mutations occur in about 40% of CRC patients. Inhibition of the EGFR pathway by cetuximab or other EGFR inhibitors may induce tumor regression, but always active mutant KRAS has been shown to confer resistance to cetuximab. Thus, the mutation status of KRAS is an important indicator of the benefits of cetuximab treatment, and cetuximab is currently used in patients with wild-type KRAS. However, almost half of wild-type KRAS patients do not respond to treatment with cetuximab. On the other hand Some patients with KRAS mutations could still respond to treatment with cetuximab. These results indicate that KRAS mutation alone is not sufficient to predict cetuximab reactivity.

In addition to KRAS, other predictive markers correlated with the response rate of cetuximab therapy are being studied. For example, downstream signaling molecules in the EGFR pathway, including BRAF, PI3K and PTEN, are known to predict negative outcomes of anti-EGFR target therapy. However, the signal molecules are not consistently associated with the response to cetuximab (De Roock et al ., Lancet Oncol . 12 (6): 594-603, 2011; Lech et al ., World J. Gastroenterol . 22 5): 1745-1755, 2016). Therefore, to improve responsiveness to treatment with cetuximab, other predictive markers need to be identified, and furthermore, drug combinations that can overcome cetuximab resistance need to be found.

To date, a method of administering a conjugate of an anti-EGFR antibody and a cytotoxic agent as a therapeutic method for cancer resistant to EGFR target therapy has been proposed (US9233171B1).

However, although the prior art promotes the death of resistant colon cancer cells and head and neck cancer cells against EGFR inhibitors, the combined cytotoxic agent maytansinoid itself also has cytotoxicity against these cancer cells And it appears to alleviate the resistance against EGFR inhibitor resistant cancer as well as other anticancer resistant cancer, and it is difficult to completely overcome the resistance to EGFR inhibitor.

Disclosure of Invention Technical Problem [8] Accordingly, 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 EGFR inhibitor-resistant cancer therapeutic agent and a screening method for the therapeutic agent. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention there is provided a pharmaceutical composition comprising: i) GNB5 inhibitors and EGFR inhibitors; Or ii) a drug conjugate wherein the GNB5 inhibitor is linked to a covalent bond to the EGFR inhibitor as an active ingredient.

According to another aspect of the present invention, there is provided a method for producing a test compound, comprising: reacting a test compound or a natural substance with GNB5; And selecting a test compound or a natural substance that specifically binds to GNB5. The present invention also provides a method for screening a candidate compound for resistance cancer treatment for an EGFR inhibitor.

According to another aspect of the present invention, there is provided a method for treating GNB5 comprising: treating a cell expressing GNB5 with a test compound or a natural product; There is provided a screening method for a cancer resistant therapeutic agent candidate for an EGFR inhibitor, comprising the step of screening a test compound or natural product significantly inhibiting the expression of GNB5 as compared with a control without any treatment.

According to another aspect of the present invention, there is provided a method for producing a compound, comprising the steps of: treating a compound or natural product with a composition comprising GNB5 and a pair protein interacting with GNB5; There is provided a method for screening a candidate for a resistance cancer therapeutic agent for an EGFR inhibitor, comprising the step of screening a test compound or a natural substance significantly inhibiting the interaction of GNB5 and the parent protein, as compared to a control not subjected to any treatment.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising: i) a GNB5 inhibitor and an EGFR inhibitor; Or ii) administering a drug conjugate wherein the GNB5 inhibitor is linked to a covalent bond to the EGFR inhibitor, to a patient suffering from a resistant cancer against an EGFR inhibitor.

According to another aspect of the present invention, there is provided a method of treating cancer, comprising: separating cancer cells from cancer patients; Treating the cancer cells with an EGFR inhibitor; Determining whether the cancer cell is resistant to the EGFR inhibitor; And, when it is determined that the cancer cell is resistant to the EGFR inhibitor, administering a combination of the GNB5 inhibitor and the EGFR inhibitor to the cancer patient in combination with an EGFR inhibitor.

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the steps of: measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient; And predicting that the cancer patient exhibiting a higher level of expression of GNB5 than normal levels has resistance to the EGFR inhibitor.

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the steps of: measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient; Selecting a cancer patient whose expression level of GNB5 is expressed higher than a normal level; And administering an EGFR inhibitor and a GNB5 inhibitor simultaneously to cancer patients screened to have a high expression level of GNB5.

According to another aspect of the present invention, there is provided a method for identifying a test compound, comprising: reacting a test compound with GNB5 and EGFR, respectively; And selecting a test compound that binds to both the active site of GNB5 and the EGFR. The present invention also provides a screening method for a candidate therapeutic substance for an EGFR inhibitor resistant cancer.

According to another aspect of the present invention, there is provided a method of treating a cell, comprising the steps of: treating a test compound with cells expressing GNB5 and EGFR; And selecting a test compound that significantly inhibits the expression or activity of GNB5 and EGFR at the same time as compared to a control group that has not been subjected to any treatment.

According to one embodiment of the present invention as described above, the treatment efficiency of the resistant intestine cancer against the EGFR inhibitor can be drastically increased. Of course, the scope of the present invention is not limited by these effects.

Figure 1 shows the process of searching for potential targets to overcome cetuximab resistance. (A) Data set Heat map of genes differentially expressed between cetuximab-resistant and cetuximab-resistant patients in GSE5851. The hue scale shows a multiple of expression relative to the intermediate expression level of each gene in all patient samples, UP and DOWN Show upregulated genes and downregulated genes in cetuximab resistant patients, respectively. (B) A graph showing expression patterns of eight genes continuously up-regulated from the cetuximab-resistant cell line of the data set GSE59857 compared to the susceptible cells. (C) A graph showing the expression levels of eight genes in cetuximab-resistant cell line NCI-H508 and cetuximab-resistant cell lines HCT116, HT29 and SW48 by qRT-PCR.
FIG. 2 shows the results of the reaction of cisplatin with KRAS wild type CRC cells by potential target knockdown, showing that (A) DUSP4, (B) ETV5, (C) GNB5, (D) NT5E and (E) PHLDA1 shRNAs FIG. 5 is a graph showing the results of cell death analysis. FIG. Each target gene was transiently knocked down in SW48 cells. Target gene-knockdown SW48 cells were cultured in 96-well plates for 24 hours (1 x 10 ⁴ cells / well) and treated with cetuximab (100 μg / mL) for 72 hours. The results are expressed as mean ㅁ standard deviation (error bars) (n = 3). The knockdown efficiency of the target gene was confirmed by Western blot or RT-PCR. β-actin was used as a loading control.
FIG. 3 shows the results of the reaction of erlotinib of KRAS wild type CRC cells with potential target knockdown by (A) DUSP4, (B) ETV5, (C) GNB5, (D) NT5E and (E) PHLDA1 shRNAs transfection FIG. 5 is a graph showing the results of cell death analysis. FIG. Each target gene was transiently knocked down in SW48 cells. Target gene-knockdown SW48 cells were cultured in 96-well plates for 24 hours (1 × 10 ⁴ cells / well) and treated with erlotinib (10 μM) for 72 hours.
FIG. 4 shows the results of (A) DUSP4, (B) ETV5, (C) GNB5, (D) NT5E, and (E) PHLDA1 shRNAs transfection in response to cetuximab of KRAS mutant CRC cells by potential target knockdown Lt; RTI ID = 0.0 > 1 < / RTI > Each target gene was transiently knocked down in HCT116 cells. Target gene-knockdown HCT116 cells were cultured in 96-well plates for 24 hours (0.7 × 10 ⁴ cells / well) and treated with cetuximab (100 μg / mL) for 72 hours. The results were expressed as mean ㅁ standard deviation ) (N = 3). The knockdown efficiency of the target gene was confirmed by RT-PCR. GAPDH was used as a loading control.
Figure 5 is a series of photographs showing that cetuximab-induced cytotoxicity is enhanced in HCT116 cells by GNB5 knockdown. To analyze cell death, 1 ㎍ / mL of propidium iodide (PI) was added to the cells, and fluorescence staining images were obtained after 72 hours of treatment with cetuximab.
Figure 6 shows the effect of GNB5 expression and GNB5 knockdown in the KRAS mutant cell line. (A) A graph showing the degree of GNB5 expression in KRAS wild type NCI-H508 cell line and cetuximab resistant cell line with other KRAS mutants. (B, C) Data set GSE59857 (B) and Cancer Cell Line Encyclopedia A graph showing the degree of GNB5 expression in the corresponding cell line, (D) a graph showing the cell viability of cetuximab and GNB5 knockdown shRNA in SW620 cells (left panel), and a GNB5 knockdown GNB5 mRNA level With the graph showing the results (right panel), the result is similar to that of FIG. 4C.
FIG. 7 shows the results of transfection of KRAS mutant CRC cells with erlotinib by potential knockdown in the presence of (A) DUSP4, (B) ETV5, (C) GNB5, (D) NT5E, and (E) PHLDA1 shRNAs FIG. 5 is a graph showing the results of cell death analysis. FIG. Each target gene was transiently knocked down in HCT116 cells. Target gene-knockdown HCT116 cells were cultured in 96-well plates for 24 hours (0.7 × 10 ⁴ cells / well) and treated with erlotinib (10 μM) for 72 hours.
Figure 8 shows the characteristics of GNB5 in colorectal cancer. (A) a graph showing the frequency of GNB5 gene modification in the CRC of cBioPortal, (B) a graph comparing GNB5 expression levels in CRC and normal adjacent mucosa, (C) the overall survival curves of CRC patients classified as GNB5 expression levels D) Normalized concentration scores (NES) and FDR-corrected P -values are presented in the GNB5 gene set enrichment assay (GSEA).
Fig. 9 shows the result of analysis of the concentration of the GNB5 gene group. The GNB5 expression data from the TCGA and GO gene sets, the carcinogenic gene sets and the gene markers of MSIGDB were used in the GSEA and the positive or negative NES and FDR-corrected P-values are presented.
Figure 10 shows the effect of cetuximab treatment on Akt and ERK signaling. (A) A photograph showing the result of Western blot analysis performed after NCI-H508 and SW48 cells were treated with 100 μg / mL of cetuximab for 1 hour, (B) for 24 hours, harvested and dissolved, (C) (D) Quantification of the expression level of each cell line at 0 hour before treatment with cetuximab, and a graph showing normalized Akt and ERK activity, respectively, by RT-PCR analysis of GNB5 expression level in H508 and SW48 cells.
Figure 11 shows the effect of GNB5 overexpression in cetuximab sensitive cells. (A) A photograph showing Western blot analysis results using anti-phosphorylated-Akt, anti-phosphorylated-ERK and anti-GNB5 antibodies against GNF5 overexpressing NCI-H508 cell lysates. And used as a control. (B) A graph showing the cell viability analysis results of the control or NCI-H508 cells expressing GNB5, the results being expressed as mean ㅁ standard deviation (error bars) (n = 3). (C) GNB5 and then inoculated over-expressing and control NCI-H508 cells at a density of 6-well plates in ² × 10 2 cells / well, the cells until small colonies to be clearly formed for 14 days growth, colonies A photograph showing the result of staining with crystal violet solution for 1 hour (top) and a graph showing the absorbance of the eluate after eluting the cell-bound dye with 1% SDS solution at 590 nm (bottom).
12 is a graph showing the results of analysis of GNB5 expression in GSE59857 through one-way dual sample t-test. S denotes a cetuximab sensitive cells (n = 10), R_WT or R_MT is (P - value <0.05), indicating the expression and each GNB5, KRAS wild-type (n = 57) and mutant-type (n = 69) of cetuximab resistant cells .
Figure 13 shows a network model of GNB5 for cetuximab resistance. (A) KRAS wild type and (B) KRAS mutation, CTX represents cetuximab, GNB5 O / E represents GNB5 overexpression, and GNB5 K / D represents GNB5 knockdown. The simulation time is expressed in arbitrary units (au), and the gray area of the shade in survival represents the boundary between sensitive and resistive reactions. (C) Represents the cetuximab resistance for the proposed GNB5 network.
Figure 14 shows the effect of GNB knockdown on Akt and ERK signaling in cetuximab treatment in SW620 cells. (A) Western blot analysis showing the levels of Akt and ERK phosphorylation in SW620 cells with KRAS mutation after knockdown and cetuximab treatment of GNB5, (B) time-dynamics graphs quantifying and normalizing Akt and ERK signals, (C) And the corresponding simulation curve for the ERK activity (CTX or GNB5 K / D represents the cetuximab treatment or knockdown condition of GNB5 respectively and the initial state of CTX + GNB5 K / D represents the normal state at the state of GNB5 K / D of the middle panel of FIG. (D) cetuximab, GNB5 knockdown, and a graph showing a comparison of Akt and ERK activity between simulation results and actual experimental results in SW620 cells after 2 hours of combined treatment.
15 shows the results of the parametric perturbation analysis of the GNB5 network model. The GNB5 network was simulated with a random arbitrary perturbation of nominal parameter values in the range of 0 to 50% and the change in survival rate was less than or equal to 0.5 at cetuximab treatment in the KRAS wild type and in combination with GNB5 knockdown and cetuximab in the KRAS mutation And showed sensitivity to cetuximab, while in other cases it was 0.5 or more.

Definition of Terms:

The term " epidermal growth factor receptor " (EGFR), as used herein, is a transmembrane protein that is a receptor for an epithelial growth factor family protein, an extracellular ligand. Mutations that affect the expression or activity of EGFR in a large number of cancers can cause the development and progression of cancer. Accordingly, anticancer agents targeting EGFR have been developed. Such anticancer agents include cetuximab (trade name: Erbitux ^® ), which is an antagonistic antibody targeting EGFR.

As used herein, the term "GNB5 (guanine nucleotide binding protein (G protein), beta 5)" is a heterotrimer protein, which is a guanine nucleotide binding protein that mediates signals between receptor and effector proteins Known as an important regulator of the alpha subunit, as well as specific signaling receptors and effectors as beta subunits, one of the three subunits (alpha, beta, and gamma subunits) Lt; RTI ID = 0.0 &gt; transcriptional &lt; / RTI &gt; GNB5 is known to interact with the G-protein signaling 7 (RGS7) and the G-protein gamma subunit 13 (GNG13), and is overexpressed in some cancer cells However, the roles of these markers in cancer are not well known except for their use as markers.

As used herein, 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.

As used herein, the term " 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).

As used herein, the term " F (ab &apos;) 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 '.

The term " Fab &quot;, as used herein, 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.

As used herein, the term " scFv " refers to a single chain fragment variable, a recombinant antibody fragment in which the variable regions (V _H and V _L ) of a Fab of an antibody are produced in a single strand using a linker do.

As used herein, the term " sdAb (single doamain antibody) " is an antibody fragment consisting of 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.

As used herein, the term " 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 non-antibody-derived protein scaffolds such as antibody fragments (V _H H, V _NAR, etc.) derived from camelia or cartilaginous fish, or nanobody, monobody, variable lymphocyte receptor (VLR).

As used herein, the term "structure-based drug design" is used to design a new active substance using computer modeling based on stereostructure and interaction information of the receptor protein and drug complex that are targets of the new drug . Since it is possible to more efficiently perform the operations from the search for the lead compound, which can be referred to as a drug candidate compound, to the optimization using the three-dimensional stereostructure information of the receptor protein, the time required for the initial drug screening, The next step is to search for compounds that can bind to this space from the library, or actually synthesize the compounds that can bind to the space, taking into account the space in which the ligand, the drug or the transmitter, binds. There are ways to check.

As used herein, the term " active site " refers to a site where a substrate binds and a chemical reaction occurs in an enzyme, and a chemical reaction occurs between the binding site and the substrate Catalytic site, which act as an important target site for drug development.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention there is provided a pharmaceutical composition comprising: i) GNB5 inhibitors and EGFR inhibitors; Or ii) a drug conjugate wherein the GNB5 inhibitor is linked to a covalent bond to the EGFR inhibitor as an active ingredient.

In the above pharmaceutical composition, the GNB5 inhibitor may be a GNB5 activity inhibitor or a GNB5 expression inhibitor, and the GNB5 activity inhibitor may be a GNB5 antagonistic antibody or a functional fragment thereof, an antibody analogue specifically binding to GNB5, a gallein, Or GRK2i, and the GNB5 expression inhibitor may be an antisense nucleotide, siRNA, shRNA or microRNA that specifically binds to the GNB5 gene.

In this pharmaceutical composition, the resistant cancer to the EGFR inhibitor may be a mutant or non-mutated KRAS gene, the mutation may be derived from exon 2 of the KRAS gene, and the mutation occurring in the exon 2 may be 12 Th and / or 13th amino acid glycine may be substituted with another amino acid.

In the above pharmaceutical composition, the EGFR inhibitor may be an EGFR inhibitory compound or an EGFR antagonistic antibody, and the EGFR inhibitory compound may be gefitinib, erlotinib, lapatinib, vadetanib, neratinib, or osimertinib, and the EGFR antagonist may be cetuximab, pantiumumab, or necitumumab.

In the pharmaceutical composition for treating EGFR inhibitor-resistant cancer, the effective substance can be provided in the form of a single compound by covalently bonding the GNB5 inhibitor with the EGFR inhibitor. Examples of these linked by covalent bonds are well known in the art. For example, Spitzer et al. Reported that 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). In particular, the 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 -).

In the above pharmaceutical composition, the cancer resistant to the EGFR inhibitor may be colon cancer, lung cancer, pancreatic cancer or head and neck cancer.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, It can have one formulation.

The pharmaceutical composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered intravenously, intranasally, intramuscularly, intraperitoneally, intrathecally, intraspinally, subcutaneously, intradermally Intraperitoneal, intrathecal, intrathecal, intracerebral, transdermal, and the like.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

According to another aspect of the present invention, there is provided a method for producing a test compound, comprising: reacting a test compound or a natural substance with GNB5; And selecting a test compound or a natural substance that specifically binds to GNB5. The present invention also provides a method for screening a candidate compound for resistance cancer treatment for an EGFR inhibitor.

In the screening method, the specific binding of GNB5 to the test compound or the natural product is determined by radioisotope labeling-using cell-affinity chromatography, drug affinity responsive target stability (DARTS) Differential scanning light scattering (DSLS) analysis, differential scanning fluorometry (DSF), or differential emissivity capillary behavior analysis of ligands (Differential Scanning Calorimetry) radial capillary action of ligand assay, DraCALA).

The screening method may further comprise the step of treating the EGFR inhibitor resistant cancer cell with the EGFR inhibitor to determine whether the EGFR inhibitor resistance is reduced by treating the selected test compound or natural product with the EGFR inhibitor resistant cancer cell.

According to another aspect of the present invention, there is provided a method for treating GNB5 comprising: treating a cell expressing GNB5 with a test compound or a natural product; And selecting a test compound or a natural substance that significantly inhibits the expression of GNB5 as compared with a control group to which nothing is treated.

In the above screening method, the expression of GNB5 can be performed by measuring the protein level of GNB5 or the expression level of mRNA encoding GNB5, and the protein level of GNB5 can be determined by measuring the functionalities of the antibody or antibody specifically binding to GNB5 Can be measured by Western blot analysis using a fragment, protein immunoprecipitation, ELISA, or RIA, or analysis based on mass or charge of proteins such as HPLC, LC / MS, or mass spectrometry, Expression levels of mRNA can be performed through Northern blot analysis, quantitative PCR, or microarray analysis. For example, an antibody specifically binding to GNB5 may be a commercial antibody (ab185206, ab116705, ab85214, etc.) sold by Abcam. Meanwhile, the expression level of the mRNA encoding GNB5 is determined by RT-PCR using a forward primer consisting of the nucleic acid sequence shown in SEQ ID NO: 7 and a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 8, more preferably RT -PCR. &Lt; / RTI &gt;

The screening method may further comprise the step of treating the EGFR inhibitor resistant cancer cell with the EGFR inhibitor to determine whether the EGFR inhibitor resistance is reduced by treating the selected test compound or natural product with the EGFR inhibitor resistant cancer cell.

According to another aspect of the present invention, there is provided a method for producing a compound, comprising the steps of: treating a compound or natural product with a composition comprising GNB5 and a pair protein interacting with GNB5; And selecting a test compound or a natural substance that significantly inhibits the interaction of GNB5 and the parent protein, as compared to a control without any treatment, and a method for screening a candidate for a resistance cancer therapeutic agent for an EGFR inhibitor.

(G protein), gamma 2 (G protein), RGS11 (regulator of G-protein signaling 11), GNG2 (guanine nucleotide binding protein (Regulator of G-protein signaling 6), RGS7 (regulator of G-protein signaling 7), RGS7BP (regulator of G-protein signaling 7 binding protein), DRD2 (dopamine receptor D2) (COP5 signalosome subunit 5), CCT3 (chaperonin containing TCP1, subunit 3), RGS9 (regulator of G protein signaling 9), GNG5 (guanine nucleotide binding protein G protein 5), CDKN1A inhibitor 1A), MTOR (mechanistic target of rapamycin), CCT2 (chaperonin containing TCP1, subunit 2), PRPF40B (PRP40 pre-mRNA processing factor 40 homolog B), CUL4A (cullin 4A), PFDN5 (prefoldin subunit 5) t-complex 1), GIT11 (heterotrimeric G protein subunit Git11), CCT6B (chaperonin containing TCP1, subunit 6B), USP38 (ubiquitin specific peptidase 38), R (APP), amyloid beta (A4) precursor protein, PA1 (replication protein A1), ANXA7 (annexin A7), E2F2 (E2F transcription factor 2), PSMD4 (proteasome, prosome, macropain) 26S subunit, MVD (mevalonate (diphospho) decarboxylase), CCT5 (chaperonin containing TCP1, subunit 5), GNG4 [guanine nucleotide binding protein (G protein), gamma 4], MCM2 (minichromosome maintenance complex component 2), BNIP3L (BCL2 / adenovirus E1B 19kDa interacting protein 3-like, CSNK2B (casein kinase 2, beta polypeptide), ELAV like binding protein 1 (ELAVL1), TSC22D1 (TSC22 domain family, member 1), or CCT7 (chaperonin containing TCP1, subunit 7) , Interaction between GNB5 and the parent protein is determined by yeast two hybrid analysis, fluorescence resonance energy transfer (FRET) analysis, affinity capture, protein fragment complementarity analysis (protein- fragment complementation assay (PCA), immunocoprecipitation (IP) Through the seating or surface plasmon resonance analysis (surface plasmon resonance assay) it may be analyzed.

The screening method may further comprise the step of treating the EGFR inhibitor resistant cancer cell with the EGFR inhibitor to determine whether the EGFR inhibitor resistance is reduced by treating the selected test compound or natural product with the EGFR inhibitor resistant cancer cell.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising: i) a GNB5 inhibitor and an EGFR inhibitor; Or ii) administering a drug conjugate wherein the GNB5 inhibitor is linked to a covalent bond to the EGFR inhibitor, to a patient suffering from a resistant cancer against an EGFR inhibitor.

According to another aspect of the present invention, there is provided a method of treating cancer, comprising: separating cancer cells from cancer patients; Treating the cancer cells with an EGFR inhibitor; Determining whether the cancer cell is resistant to the EGFR inhibitor; And, when it is determined that the cancer cell is resistant to the EGFR inhibitor, administering a combination of the GNB5 inhibitor and the EGFR inhibitor to the cancer patient in combination with an EGFR inhibitor.

According to another aspect of the present invention, there is provided a method of treating a cell, comprising the steps of: treating a test compound with cells expressing GNB5 and EGFR; And selecting a test compound that significantly inhibits the expression or activity of GNB5 and EGFR at the same time as compared to a control group that has not been subjected to any treatment.

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the steps of: measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient; And predicting that the cancer patient exhibiting a higher level of expression of GNB5 than normal levels has resistance to the EGFR inhibitor.

According to another aspect of the present invention, there is provided a method for diagnosing cancer, comprising the steps of: measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient; Selecting a cancer patient whose expression level of GNB5 is expressed higher than a normal level; And administering an EGFR inhibitor and a GNB5 inhibitor simultaneously to cancer patients screened to have a high expression level of GNB5.

According to another aspect of the present invention, there is provided a method for identifying a test compound, comprising: reacting a test compound with GNB5 and EGFR, respectively; And selecting a test compound that binds to both the active site of GNB5 and the EGFR. The present invention also provides a screening method for a candidate therapeutic substance for an EGFR inhibitor resistant cancer.

The method comprises the steps of: treating the selected test compound with EGFR resistant cancer cells overexpressing GNB5; And screening a test compound that inhibits the growth of EGFR resistant cancer cells.

In the above method, the test compound may be designed by a structure-based drug design. For example, the structure-based drug design uses a three-dimensional structure by computer modeling of GNB5 and EGFR to determine whether a compound of a specific structure binds to the active site of GNB5 and / or EGFR, Can be carried out by changing the structure of the compound through 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, .

Whether or not the test compound inhibits its activity by binding to the actual GNB5 can be confirmed using the above-described various GNB5-small compound interaction assay method and / or GNP5-interacting paired protein interaction assay method have.

Similarly, whether or not the test compound inhibits the activity of EGFR can be analyzed using a method similar to that for analyzing whether the test compound inhibits the activity of GNB5. For example, whether or not the EGFR binds to a specific test compound is determined by radioimmunoassay labeling-affinity chromatography-affinity chromatography, drug affinity-responsive target stability (DARTS), stability- (DSF), differential scanning fluorometry (DSF), or differential radial capillary action of ligand assay (DSF), or differential radial capillary action of ligand , &Lt; / RTI &gt; DraCALA).

On the other hand, whether or not the test compound inhibits the activity of EGFR can be performed by examining whether the interaction of the EGFR and the mating protein interacting with EGFR in the upper or lower part is inhibited in the test compound treatment, Androgen receptor, ARF4, CAV1, CAV3, CBL, CBLB, CBLC, CD44, CDC25A, CRK, CTNNB1, DCN, EGF, Grb14, JAK2, MUC1, NCK1, NCK2, PKCα, PLCG1, PLSCR1, PTPN1, PTPN11, PTPRK, PTPRK , Sh2D3A, SH3KBP1, SHC1, SOS1, Src, STAT1, STAT3, Stat5A, UBC, WAS, TACC3, Skap2, Ddx17, Dok3, Cnk2, Amph, Lzts2, Usp33, Diaph1, Rim2, or Mbip.

The present inventors identified five potential target genes (DUSP4, ETV5, GNB5, NT5E, and PHLDA1) for cetuximab-resistant CRC through gene expression data analysis and cell line experiments. Knockdown of these 5 genes in KRAS wild type cells increased susceptibility to cetuximab. In addition, we confirmed that knockdown of GNB5 increases cetuximab susceptibility in KRAS mutant cells. The present inventors further investigated the role of GNB5 in cancer progression and found that GNB5 contributes to cetuximab resistance by predominantly regulating Akt signaling pathway. Our findings suggest that GNB5 may be a promising target for combination therapy with cetuximab in cetuximab resistant cancers, regardless of KRAS mutations.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

General method

Gene expression data collection and analysis:

Gene expression data for CRC patients who received Cetuximab monotherapy (GSE5851) were obtained from the Gene Expression Omnibus (GEO, //www.ncbi.nlm.nih.gov/geo). Differentially expressed genes (DEG) between cetuximab-sensitive and resistant patients were identified using the Benjamini-Hochberg step-down FDR algorithm. The algorithm adjusts the P -value to reflect the effects of various tests. The heat map of differentially expressed genes was generated by a multiple change of each gene to the average expression level. Gene expression data of human CRC cell line using cetuximab treatment (GSE59857) was also obtained from GEO. The gene expression data pairs of CRC and normal adjacent mucosa were obtained from TCGA and compared using the Wilcoxon rank sum test.

Gene group concentration analysis:

A set of input data for gene set enrichment assay (GSEA) analysis was prepared as follows: (1) RNA-seq data from CRC patients were obtained from TCGA. (2) The phenotype marker is determined based on the median value of GNB5 expression. (3) A gene ontology (GO) gene set, a carcinogenic gene set and an imprinted gene set were downloaded from MSigDB (// www.broadinstitute.org/gsea/msigdb). Other parameters were set to default values. The GSEA analysis was performed using a javaGSEA Desktop Application with 3,000 permutations.

Clinical data analysis:

The overall survival rate of CRC patients was obtained from TCGA. Kaplan-Meier plots of overall survival were generated and compared by log rankings using Survival package in R. Expression levels of GNB5 were differentiated high or low on the basis of median values.

Mathematical modeling:

Ordinary differential equations (ODEs) of the network model are mainly based on Michaelis-Menten dynamics:

(In the above equation, <molecule> a or <molecule> i represents the concentration of the active or inactive form of each <molecule>, and the total concentration of the two types of molecules is set to <molecule> tot).

The inhibition of EGFR by cetuximab was modeled by competitive inhibition kinetics. Survival variables are calculated as a linear combination of ERKa and AKTa, yielding quantities with arbitrary units. The KRAS mutation of this model was used by reduced degradation of KRAS. It is important to note that the network model is structured to describe the mechanism in a qualitative way without rigorously reproducing the amount of actual biochemical reaction. Nominal or conditioned values of kinetic parameters and concentrations were determined to simply indicate the causal relationship between signal molecules. All parameters used in the analysis are as described in Tables 1 and 2 below.

Cell culture:

NCI-H508, HCT116 and HEK293T cells were incubated with 10% fetal bovine serum (FBS) and antibiotics (100 units / ml penicillin, 100 ug / ml streptomycin and 0.25 ug / (Dulbecco's modified Eagle's medium, DMEM, Welgene Inc., Korea) and incubated at 37 ° C in a humidified atmosphere containing 5% CO ₂ . SW48 cells were cultured in a culture medium (Leibovitz's L15 Medium) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 units / ml penicillin, 100 占 퐂 / ml streptomycin and 0.25 占 퐂 / , Welgene Inc., Korea) and cultured at 37 ° C in a humidified atmosphere containing 5% CO ₂ .

reagent:

Cetuximab was purchased from Merck Sereno (Erbitux, Darmstadt, Germany) and dimethylsulfoxide (DMSO) and propidium iodide (PI) were purchased from Sigma-Aldrich (Saint Louis, Mo.). Erlotinib was purchased from Cell Signaling (Danvers, MA).

Differential expression genes between cetuximab sensitive and resistant patients from GSE5851 ID_REF Gene symbol Entrez ID P -value Expression 203939_at NT5E 4907 2.585E-04 UP 217999_s_at PHLDA1 22822 5.441E-04 UP 203349_s_at ETV5 2119 5.469E-04 UP 208130_s_at TBXAS1 6916 6.070E-04 UP 204014_at DUSP4 1846 6.082E-04 UP 206503_x_at PML 5371 6.199E-04 UP 202298_at NDUFA1 4694 6.531E-04 UP 219297_at WDR44 54521 6.906E-04 UP 209029_at COPS7A 50813 7.025E-04 UP 211871_x_at GNB5 10681 9.110E-04 UP 217645_at COX16 51241 9.405E-04 UP 205767_at EREG 2069 5.620E-07 UP 205239_at AREG 374 6.722E-07 DOWN 212349_at POFUT1 23509 4.923E-05 DOWN 220818_s_at TRPC4 7223 6.296E-05 DOWN 200884_at CKB 1152 1.586E-04 DOWN 220642_x_at GPR89A 51463 1.598E-04 DOWN 219615_s_at KCNK5 8645 1.729E-04 DOWN 207653_at FOXD2 2306 1.871E-04 DOWN 221027_s_at PLA2G12A 81579 2.004E-04 DOWN 211801_x_at MFN1 55669 2.791E-04 DOWN 205112_at PLCE1 51196 3.278E-04 DOWN 204379_s_at FGFR3 2261 3.578E-04 DOWN 203560_at GGH 8836 3.853E-04 DOWN 204087_s_at SLC5A6 8884 4.012E-04 DOWN 209790_s_at CASP6 839 4.239E-04 DOWN 203434_s_at MME 4311 4.465E-04 DOWN 203858_s_at COX10 1352 4.533E-04 DOWN 202925_s_at PLAGL2 5326 5.097E-04 DOWN 220166_at CNNM1 26507 6.830E-04 DOWN 203123_s_at SLC11A2 4891 7.151E-04 DOWN 206432_at HAS2 3037 7.507E-04 DOWN 203673_at TG 7038 8.322E-04 DOWN 207127_s_at HNRNPH3 3189 8.373E-04 DOWN 204936_at MAP4K2 5871 8.582E-04 DOWN 220161_s_at EPB41L4B 54566 8.952E-04 DOWN 206552_s_at TAC1 6863 9.008E-04 DOWN 218618_s_at FNDC3B 64778 9.339E-04 DOWN 213499_at CLCN2 1181 9.544E-04 DOWN 203943_at KIF3B 9371 1.027E-03 DOWN 207705_s_at NINL 22981 1.040E-03 DOWN 216760_at HRASLS2 54979 1.074E-03 DOWN

Nominal values of kinetic parameters and concentrations in ODE simulations number name Explanation value Dynamic parameter One k _One Activation of EGFR by EGF One 2 K _m1 Activation of EGFR by EGF 0.5 3 K _i1 Inhibition of EGFR by Cetuximab 0.1 4 k _d1 Basic degradation of EGFR 0.5 5 k _2a Activation of KRAS by EGFR 5 6 k _2b Activation of KRAS by GNB5 2 7 K _m2 Activation of KRAS by EGFR and GNB5 10 8 k _d2 Basic decomposition of KRAS 5 9 k ₃ Activation of MEK by KRAS 2 10 K _m3 Activation of MEK by EGFR and KRAS One 11 k _d3 Basic disassembly of MEK 0.5 12 k ₄ Activation of ERK by MEK One 13 K _m4 Activation of ERK by MEK One 14 k _d4 Basic decomposition of ERK 0.5 15 k _5a Activation of PI3K by KRAS 5 16 k _5b Activation of PI3K by GNB5 2 17 k _5c Inhibition of PI3K by ERK 10 18 K _m5 Activation of PI3K by KRAS and GNB5 10 19 K _i5 Inhibition of PI3K by ERK 10 20 k _d5 Basic decomposition of PI3K 0.5 21 k ₆ Activation of AKT by PI3K One 22 K _m6 Activation of AKT by PI3K 5 23 k _d6 Basic decomposition of AKT 0.5 24 w _ERK Linear combination weights for survival variables 0.5 25 w _AKT Linear combination weights for survival variables 0.5 density One EGFRtot Total concentration of EGFR One 2 KRAStot Total concentration of KRAS One 3 Mektot Total concentration of MEK One 4 ERKtot Total concentration of ERK One 5 PI3Ktot Total concentration of PI3K One 6 AKTtot Total concentration of AKT One 7 EGF The concentration of EGF in the control condition One 8 Cetuximab Concentration of cetuximab in the control condition 0 9 GNB5 GNB5 in control condition 0.5

Total RNA extraction, RT-PCR and qRT-PCR:

Total RNA was extracted from the cells using a PureLink ^ㄾ RNA Mini Kit (Thermo Fisher Scientific Inc., Waltham, MA) according to the manufacturer's standard protocol and DNase I (Thermo Fisher Scientific Inc.) The genomic DNA was removed. Then, reverse transcription (RT) reaction was performed using DiaStar RT kit (Solgent, Daejeon, Republic) to synthesize cDNA from total RNA. RT-PCR analysis was carried out using primers for the human genes described in Table 3 below using a PCR system (Veriti 96well Thermal Cycler, Applied Biosystems, Waltham, Mass.). Quantitative RT-PCR analysis was performed using the QuantStudio 5 real-time PCR system (Applied Biosystems) using the corresponding primers.

Primers used in PCR gene Forward primer (SEQ ID NO) The reverse primer (SEQ ID NO) COPS7A 5'-TGA TGA GTG CGG AAG TGA AG-3 '(1) 5'-TTC CGG GCT TCA GCT AAG TA-3 '(2) DUSP4 5'-TCA CGG CTC TGT TGA ATG TC-3 '(3) 5'-TCA CGG CTC TGT TGA ATG TC-3 '(4) ETV5 5'-GAC ACA GAT CTG GCT CAC GA-3 '(5) 5'-GGC ATG AAG CAC CAG GTT AT-3 '(6) GNB5 5'-GGG AAC AAA GTC CTG TGC AT-3 '(7) 5'-TAT CCA AAC CAC CAC AAGCA-3 '(8) NT5E 5'-GGC TGC TGT ATT GCC CTT TG-3 '(9) 5'-ATA CAC CAC ATG GAT TCC GCC-3 '(10) PHLDA1 5'-GGT CAA GCT CAA GGA ACT GC-3 '(11) 5'-GAT GGC CTG ACG ATT CTT GT-3 '(12) PML 5'-ACA CAA CGT GAG CTT CAT GG-3 '(13) 5'-ACA CAA CGT GAG CTT CAT GG-3 '(14) TBXAS1 5'-ATT GCA GAG ACG CTG AGG AT-3 '(15) 5'-CAC GTG GAG CAG TGT CAA CT-3 '(16) WDR44 5'-TCT CTC CTA ACC GCA AGC AT-3 '(17) 5'-TGC TAC AGT CTG CCC ATC AG-3 '(18) β-Actin 5'-AGA GCT ACG AGC TGC CTG AC-3 '(19) 5'-AGC ACT GTG TTG GCG TAC AG-3 '(20) GAPDH 5'-CGC TCT CTG CTC CTC CTG TT-3 '(21) 5'-CCA TGG TGT CTG AGC GAT GT-3 '(22)

Plasmid construction and virus production:

HEK 293T cells were transfected with related lentiviral plasmid and packaging mixtures (pLP1, pLP2 and pLP / VSVG) using Lipofectamine (Invitrogene, Carlsbad, CA) according to the manufacturer's instructions to generate lentiviral particles. For overexpression experiments, full-length human GNB5 was PCR amplified from human brain cDNA library (Clontech), ligated to pLentiM1.4 lentivirus vector, and sequenced. For the knockdown experiments, pLKO.1 lentiviral-based plasmids containing shRNAs for DUSP4, ETV5, NT5E, GNB5 and PHLDA1 (Sigma-Aldrich, USA) were used.

Cell death analysis:

PI-based assays were performed to analyze cell death. The degree of apoptosis was detected according to the manufacturer's instructions using IncuCyte ZOOM (Essen Biosciences, Ann Arbor, MI, USA). Temporally knock down or overexpressed cells were seeded in 96-well plates and cultured for 24 hours (1 x 10 ⁴ cells / well). Cells were then treated with cetuximab (Merck Sereno, USA) at the indicated concentrations for 72 hours. After seeding, cells were imaged using IncuCyte ZOOM. To assess cell death, the mean area of PI-labeled cells at each time point was determined using IncuCyte ZOOM analysis software. Images were acquired at 3 hour intervals from three separate areas per well with a 20x objective.

Colony formation assay:

NCI-H508 cells (200 cells) were seeded in 6-well plates (n = 3) and cultured at 37 占 폚 for 14 days. Cells were fixed with 3.7% PFA for 15 minutes, stained with 0.5% crystal violet solution for 1 hour, rinsed with distilled water, and air dried. The cell-bound crystal violet was dissolved in 1% SDS and the absorbance was measured at 590 nm using a UV-VIS spectrophotometer (xMark ^TM microplate spectrophotometer, Bio-Rad, USA).

Western blot analysis:

Western blotting was performed as previously described. Briefly, cells were lysed in lysis buffer (20 mM HEPES, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 ug / ml apronitin, 1 ug leupeptin, 1 mM Na ₃ VO ₄ , 1 mM NaF) . Protein bands were quantified using ImageJ (NIH, Bethesda, MD, USA).

(Cell Signaling Technology, Inc., USA), anti-DUSP4 antibody (Cell Signaling Technology, Inc., USA), anti-ERK1 / 2 antibody (Santa Cruz Biotechnology, USA), anti-ETV5 antibody (Santa Cruz Biotechnology, USA), anti-beta-actin antibody (Santa Cruz Biotechnology, USA) Labeled antibody (Merck, Billerica, USA) was used. Antibodies to phospho-Akt and phospho-ERK1 / 2 were purchased from Cell Signaling Technology. The rabbit polyclonal anti-GAPDH antibody was donated by Dr. Kwon, Ki-sun (Korea Research Institute of Bioscience and Biotechnology).

Search for compounds or natural products capable of inhibiting the function or expression of GNB5:

GNB5 is an intracellular protein that is already known to interact with various partner proteins or conjugate proteins. Thus, when a particular compound or natural product is combined with the GNB5 protein, the activity of GNB5 can be inhibited by interfering with the interaction with these paired proteins. Thus, by screening for the binding of the GNB5 protein to the test compound or the natural substance, it is possible to screen candidate therapeutic agents for cancer resistant to the EGFR inhibitor by inhibiting GNB5 activity.

For the screening of the GNB5 activity inhibitor, various protein and compound binding assays can be used. These methods include radioisotope labeling-using small molecule-affinity chromatography, drug affinity responsive target stability analysis, (DSR) analysis, differential scanning fluorometry (DSF), and differential radiation capillary (DSF) analysis of the ligand. (McFedries et al ., Chem. Biol ., 20: 667-673, 2013). In addition, the assay may be performed using a differential radial capillary action of ligand assay (DraCALA). This document is incorporated herein by reference.

In addition, for screening the activity inhibitor of GNB5, a test compound or a natural substance is treated with GNB5 and a conjugate protein-containing reaction solution known to interact with GNB5, and then the test compound or natural product . &Lt; / RTI &gt; Methods for analyzing the interaction between these proteins include yeast two hybrid analysis, immunocomplex assay, surface plasmon resonance assay, continuous affinity purification-mass spectrometry (TAP-MS), fluorescence resonance energy transfer (FRET) analysis, a protein-fragment complementation assay, a phage display, an affinity capture, a reconstituted complex analysis, etc. These methods can also be used (Rao et al ., Int. J. Proteom ., 147648, 2014, Phizicky and Fields, Microbiol . Rev. 59 (1): 94-123, 1995). These documents are also incorporated herein by reference.

On the other hand, the GNB5 expression inhibitor that inhibits GNB5 expression can be designed based on the nucleic acid sequence of the nucleic acid molecule encoding GNB5. Such GNB5 expression inhibitors include small interfering RNA (siRNA), short hairpin RNA (shRNA) targeting oligonucleotide molecules that specifically bind to nucleic acid molecules encoding GNB5 or complementary nucleic acid molecules thereof, or nucleic acid molecules that encode GNB5, Or a nucleic acid molecule that induces RNA interference phenomena such as microRNAs (miRNAs).

Example 1: Selection of candidate targets capable of overcoming cetuximab resistance

To identify potential targets to overcome resistance to cetuximab, we first analyzed the gene expression data of the Khambata-Ford data set (GSE5851) annotated in response to cetuximab monotherapy in 80 mCRC patients (resistance : n = 42, sensitivity: n = 26, medium: n = 12). We found 42 genes ( P < 0.05 with FDR correction) differentially expressed in 13,162 genes between resistant and susceptible patients (Figure 1A and Table 1). These genes consist of 11 upregulated genes and 31 downregulated genes in resistant patients. Subsequent analyzes focused on upregulated genes and confirmed target gene inhibition that could cause synergy with cetuximab therapy. To this end, the present inventors analyzed gene expression data (GSE59857) obtained by tinning the reactivity to cetuximab in an additional 155 cell lines (resistance: n = 131, sensitivity: n = 10, medium: n = 14). Eight out of twelve up-regulated genes in resistant patients were consistently up-regulated in resistant cell lines (Fig. 1B). Quantitative RT-PCR (qRT-PCR) was performed on eight up-regulated genes in cetuximab-responsive cell line (NCI-H508) and cetuximab resistant cell line (HCT116, HT29 and SW48) ). As shown in Fig. 1C, the dual specificity phosphatase 4 (DUSP4), ETV translocation variant 5, NT5E (5'-nucleotidase), GNB5 (guanine nucleotide-binding protein subunit beta-5) And the expression of PHLDA1 (Pleckstrin homology-like domain family member 1) was significantly increased.

Example 2: Correlation between inhibition of potential target gene and cetuximab reactivity recovery

The five potential target genes DUSP4, ETV5, GNB5, NT5E, and PHLDA1 have been suggested to be related to the reactivity of cetuximab in some previous studies (Khambata-Ford et al ., J. Clin. Oncol . ... 3230-3237, 2007; Cushman et al, Clin Cancer Res 21 (5):.. 1078-1086, 2015; Oliveras-Ferraros et al, J. Cell Biochem 112 (1): 10-29, 2011; Balko et al ., BMC Cancer . 9: 145, 2009), there was no experimental study of how cancer cells actually responded. Therefore, the inventors of the present invention found that the potential target gene is an effective combination target capable of inducing cetuximab responsiveness in CRC, thereby inducing RNA interference by expressing shRNA against the genes, and by the knockdown of the five genes (Fig. 2 and Fig. 5). The results showed that the cell response by each of these knockdown was different depending on the mutation of KRAS. As shown in Figure 2, knockdown for the five potential target genes increased cetuximab responsiveness in the KRAS wild type cetuximab resistant cell line, SW48. All of the knockdowns of the five potential target genes showed increased cell death relative to the control. The present inventors have also found that the susceptibility to erlotinib (FIG. 3), another known EGFR inhibitor, in knockdown of any of the five target genes is increased.

Interestingly, in the case of the KRAS mutant cell line, cetuximab-resistant cell line HCT116, it was confirmed that the knockdown of GNB5 increased susceptibility to cetuximab (Fig. 4C and Fig. 5). On the other hand, in HCT116 cells, DUSP4, ETV5, NT5E or PHLDA1 knockdown showed no difference in cetuximab reactivity with the control scrambled shRNA treated group (FIGS. 3A, 3B, 3D and 3E). In another KRAS mutant SW620 cell line showing high GNB5 expression, the combined effect of cetuximab treatment and GNB5 knockdown was confirmed (Fig. 6). In the case of GNB5 knockdown in HCT116 cells, drug susceptibility to erlotinib was also found to increase (Fig. 7C). In the case of DUSP4, ETV5, NT5E or PHLDA1 knockdown in HCT116 cells, there was no difference in erlotinib sensitivity (FIGS. 7A, 7B, 7D and 7E). These results suggest that targeting GNB5 may be a new way to overcome tolerance to cetuximab as well as other EGFR inhibitors, regardless of KRAS mutations.

Example 3: Role of GNB5 in cancer progression

The present inventors investigated the characteristics of GNB5 in a database to identify the role of GNB5 in CRC. Since biomarkers are often overexpressed or mutated in cancer cells, genetic variation of CRC was identified in cBioPortal (// cbioportal.org) (Cerami et al ., Cancer Discov. 2 (5): 401-404, 2012). As a result, there was almost no amplification, mutation or deletion of the GNB5 gene in the CRC, as shown in Fig. 8A. Thus, the present inventors investigated whether GNB5 is highly expressed in CRC using the RNA-seq data of TCGA (Cancer Genome Atlas) (Giannakis et al ., Cell reports. 15 (4): 857-865, 2016). However, despite the investigation of 50 colorectal tumors and adjacent normal mucosa, there was no difference in the expression pattern of GNB5 (Fig. 8B).

To analyze the association between GNB5 expression and clinical outcome, we analyzed the overall survival rate of 282 CRC patients in TCGA. Interestingly, high GNB5 expression was found to reduce the survival rate of CRC patients (Figure 8C). Kaplan-Meier survival curves of overall survival showed that overexpression of GNB5 was significantly correlated with poor prognosis in CRC patients. To confirm the signaling pathways regulated by GNB5, gene cluster concentration analysis (GSEA) was performed using the RNA-seq data of TCGA (n = 619). As a result, genes up-regulated by GNB5 were strongly associated with epithelial cell proliferation (Fig. 8D, left panel). The genes were also highly enriched in a set of genes associated with hypoxia and epithelial-mesenchymal (EMT) (Fig. 9). These results also suggest that the function of GNB5 may be related to the progression of cancer. Furthermore, the present inventors confirmed that GNB5 has a positive correlation with genes upregulated by MEK or Akt (Fig. 8D, center and right panel).

Example 4: Relation of GNB5 expression with resistance to cetuximab

GNB5 is a molecule downstream of the largest receptor family of G protein coupled receptors (GPCRs) mediating physiologically important signaling (Osmond et al ., Curr. Opin. Mol. Ther . 12 (3): 305 -315, 2010). One of the Gβγ subunits, GNB5, signals from the GPCR to various downstream effectors (O'Hayre et al ., Curr. Opin. Cell Biol. 27: 126-135, 2014). To explore the role of GNB5 in cetuximab resistance, we investigated whether the cell signaling pathways of MEK and Akt are related to cetuximab resistance (Fig. 10). As a result, as shown in FIG. 10, cetuximab inhibited ERK and Akt phosphorylation in cetuximab-sensitive NCI-H508 cells but not cetuximab-resistant SW48 cells. Thus, the present inventors further investigated whether the level of ERK and Akt activity could be an indicator of cetuximab resistance by manipulating the expression of GNB5. Specifically, the present inventors analyzed phosphorylation of ERK and Akt by overexpression of GNB5 in NCI-H508 cells (FIG. 11A). As a result, phosphorylation of Akt was significantly increased by overexpression of GNB5, whereas phosphorylation of ERK was moderate, as shown in Fig. 11A. From these results, the present inventors confirmed that GNB5 contributes to cetuximab resistance by acting predominantly on Akt signaling rather than ERK signaling. We further investigated the role of GNB5 in cetuximab treatment (Figure 11B). As a result, it was confirmed that cetuximab treatment of GNB5 overexpressing NCI-H508 cells significantly increased cell viability as compared with the control group, as shown in Fig. 11B. In addition, colony formation analysis confirmed that GNB5 overexpressing cells were more competent for colony formation than the control (Fig. 11C). These results suggest that GNB5 may play a role in increasing cell proliferation and resistance to cetuximab by predominantly regulating Akt activity.

Example 5: Relationship between GNB5 expression and cetuximab responsiveness and KRAS mutation

Based on the above results, the present inventors investigated the relationship between GNB5 expression, cetuximab reactivity and KRAS mutation. Specifically, GNB5 expression of GES59757 was analyzed by unidirectional double-sample t-test (Figure 12). As a result, as shown in Fig. 12, the expression pattern of GNB5 appeared independent of the mutation of KRAS. Thus, GNB5 inhibitors may be effective in combination with EGFR inhibitors, independent of KRAS mutations.

Example 6: Mathematical model for identifying cetuximab resistance by GNB5

To clarify the basic mechanism of GNB5, a mathematical model of the EGFR-GNB5 signaling network was constructed as described above. In the control network model of the inventors, the standard RAS-MEK-ERK and PI3K-Akt route, as well as interaction with GNB5 KRAS and PI3K was incorporated, including the cross-regulation between ERK and PI3K (Won et al., J. Mol. Cell Biol. 4 (3): 153-163, 2012). All specifications of the network model were mathematically described using Ordinary Differential Equations (ODEs). The magnification changes of ERK and Akt activity were calculated based on activity levels under control conditions. To describe the range of cell viability, the survival parameter was defined as a linear combination of ERK and Akt activity. We have determined the cetuximab responsiveness of the network model according to one embodiment of the present invention, depending on the value of the survival variable. For the KRAS wild type, the cell viability of the network model was reduced by cetuximab treatment (Fig. 13A, left panel). As experimentally observed in Figure 9A, overexpression of GNB5 mainly increased Akt activity (Figure 13A, middle panel) and then led to cetuximab resistance (Figure 13A, right panel). KRAS mutation resulted in cetuximab resistance (Fig. 13B, left panel). The knockdown of GNB5 alone did not significantly change the cell viability of the network model (Fig. 13B, middle panel), but the cell viability was effectively reduced by treating cetuximab with susceptibility to cetuximab (Fig. 12B, panel). The kinetics of the network model according to one embodiment of the present invention was confirmed by measuring changes in Akt and ERK phosphorylation levels over time in KRAS mutant SW620 cells (Figure 14A) (Figs. 14B and 14C). &Lt; / RTI &gt; Simulation results for the KRAS mutant model generally showed similar trends to those of SW620 cells (Fig. 12B and Fig. 14D). As a result of the robustness test on the above model, we can see that the conclusions presented in the model are robust against random parameter perturbations (Fig. 15). Taken together, we have demonstrated that GNB5 confers resistance to cetuximab by governing Akt signaling as shown in Figure 11C, and thus is a promising target molecule to overcome cetuximab resistance.

In the present invention, the main mechanism of drug resistance by GNB5 is described as Akt and its activity. However, in addition to this, another mechanism related to cetuximab resistance of GNB5 is immunological effect, that is, overexpression of GNB5 induces antibody-dependent cellular cytotoxicity ADCC). &Lt; / RTI &gt;

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Korea Advanced Institute of Science and Technology <120> An anticancer agent for treating cancers resistant to EGFR          inhibitor <130> PD17-5601 <150> KR 10-2017-0146518 <151> 2017-11-06 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for COPS7A <400> 1 tgatgagtgc ggaagtgaag 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for COPS7A <400> 2 ttccgggctt cagctaagta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for DUSP4 <400> 3 tcacggctct gttgaatgtc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for DUSP4 <400> 4 tcacggctct gttgaatgtc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for ETV5 <400> 5 gacacagatc tggctcacga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for ETV5 <400> 6 ggcatgaagc accaggttat 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for GNB5 <400> 7 gggaacaaag tcctgtgcat 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for GNB5 <400> 8 tatccaaacc accacaagca 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for NT5E <400> 9 ggctgctgta ttgccctttg 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for NT5E <400> 10 atacaccaca tggattccgc c 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for PHLDA1 <400> 11 ggtcaagctc aaggaactgc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for PHLDA1 <400> 12 gatggcctga cgattcttgt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for PML <400> 13 acacaacgtg agcttcatgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for PML <400> 14 acacaacgtg agcttcatgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for TBXAS1 <400> 15 attgcagaga cgctgaggat 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for TBXAS1 <400> 16 cacgtggagc agtgtcaact 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for WDR44 <400> 17 tctctcctaa ccgcaagcat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for WDR44 <400> 18 tgctacagtc tgcccatcag 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for beta-actin <400> 19 agagctacga gctgcctgac 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for beta-actin <400> 20 agcactgtgt tggcgtacag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer for GAPDH <400> 21 cgctctctgc tcctcctgtt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer for GAPDH <400> 22 ccatggtgtc tgagcgatgt 20

Claims (20)

I) GNB5 inhibitors and EGFR inhibitors; or
Ii) a pharmaceutical composition for treating a resistant cancer against an EGFR inhibitor, wherein said GNB5 inhibitor comprises a drug conjugate linked to a covalent bond to said EGFR inhibitor as an active ingredient. The method according to claim 1,
Wherein said GNB5 inhibitor is a GNB5 activity inhibitor or a GNB5 expression inhibitor. 3. The method of claim 2,
Wherein said GNB5 activity inhibitor is a GNB5 antagonistic antibody or a functional fragment thereof, an antibody analogue that specifically binds GNB5, gallein or GRK2i. 3. The method of claim 2,
Wherein the GNB5 expression inhibitor is an antisense nucleotide, siRNA, shRNA or microRNA that specifically binds to the GNB5 gene. The method according to claim 1,
Wherein the cancer resistant to the EGFR inhibitor is a cancer that has or does not mutate in the KRAS gene. 6. The method of claim 5,
Wherein said mutation occurs in exon 2 of the KRAS gene. The method according to claim 1,
Wherein the resistant cancer against EGFR is colon cancer, lung cancer, pancreatic cancer or head and neck cancer. The method according to claim 1,
Wherein the EGFR inhibitor is an EGFR inhibitory compound, an EGFR antagonist or a functional fragment thereof or an antibody analogue that specifically binds to an EGFR. 9. The method of claim 8,
Wherein said EGFR inhibiting compound is gefitinib, erlotinib, lapatinib, vadetanib, neratinib, or osimertinib. 9. The method of claim 8,
Wherein said EGFR antagonist antibody is cetuximab, pantiumumab, or necitumumab. Reacting the test compound or the natural substance with GNB5; And
A method for screening a candidate substance for resistance cancer treatment for an EGFR inhibitor, comprising the step of selecting a test compound or natural product specifically binding to GNB5 Treating the cells expressing GNB5 with a test compound or a natural product; And
Selecting a test compound or a natural substance that significantly inhibits the expression of GNB5 compared with a control group in which no treatment is performed; and selecting a candidate compound for a cancer-treating agent for an EGFR inhibitor. Treating the test compound or the natural product with a composition comprising GNB5 and a pair protein that interacts with GNB5; And
Selecting a test compound or a natural substance that significantly inhibits the interaction of GNB5 and the parent protein, as compared to a control group to which no treatment is applied. Measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient;
And predicting the cancer patient having an expression level of GNB5 higher than normal level as having a resistance to the EGFR inhibitor. A pharmaceutically effective amount of i) GNB5 inhibitors and EGFR inhibitors; Or ii) administering the drug conjugate wherein the GNB5 inhibitor is linked to a covalent bond to the EGFR inhibitor, to a patient suffering from an EGFR inhibitor resistant cancer. Measuring the degree of expression of GNB5 from a tissue sample or a body fluid sample obtained from a cancer patient;
Selecting a cancer patient whose expression level of GNB5 is expressed higher than a normal level; And
And administering EGFR inhibitor and GNB5 inhibitor at the same time to a cancer patient selected as having a high expression level of GNB5. Reacting the test compound with GNB5 and EGFR, respectively; And
Selecting a test compound that binds to both the GNB5 and the active site of the EGFR. &Lt; Desc / Clms Page number 24 &gt; 18. The method of claim 17,
Wherein the test compound is designed by a structure-based drug design. &Lt; Desc / Clms Page number 19 &gt; 18. The method of claim 17,
Treating the selected test compound with EGFR resistant cancer cells overexpressing GNB5; And
A method of screening a therapeutic candidate compound for an EGFR inhibitor resistant cancer, further comprising the step of selecting a test compound that inhibits the growth of EGFR resistant cancer cells. Treating the test compound with cells expressing GNB5 and EGFR; And
Selecting a test compound that significantly inhibits the expression or activity of GNB5 and EGFR at the same time as compared with a control group that has not been treated with any one.

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