Pharmaceutical composition for sensitizing EGFR-TKI drug-resistant tumor treatment and application thereof

Abstract

The invention provides a pharmaceutical composition for sensitizing EGFR-TKI drug-resistant tumor treatment and application of the pharmaceutical composition. The pharmaceutical composition comprises a) a therapeutically effective amount of decitabine and b) a therapeutically effective amount of an EGFR tyrosine kinase inhibitor. The combined treatment scheme provided by the invention directly aims at the EGFR-TKI drug-resistant tumor problem, has a clear treatment target spot and relatively high clinical transformation potential, provides a new treatment choice for patients, and provides a theoretical basis and a practical reference for formulating a future individualized treatment scheme.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a pharmaceutical composition for sensitizing the treatment of EGFR-TKI-resistant tumors and its applications. Background Art

[0002] Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer. Among them, epidermal growth factor receptor (EGFR) gene mutations, as key driving factors, have a detection rate as high as 30%-40% in the Asian population, especially exon 19 deletions and exon 21 L858R point mutations are the most common. For EGFR-sensitive mutations, the first-generation EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, and the second-generation TKIs such as afatinib, block downstream signal transduction by competitively inhibiting the ATP-binding domain, significantly prolonging the progression-free survival (PFS) of patients. However, the efficacy of such drugs is generally lost due to the T790M resistance mutation after 9-14 months of treatment. As a third-generation EGFR-TKI, osimertinib can effectively inhibit the T790M mutation and showed a median PFS of 18.9 months superior to that of the first-generation drugs in the FLAURA clinical trial, becoming the new standard for first-line treatment. Nevertheless, the long-term application of osimertinib still faces the severe challenge of acquired resistance. Approximately 50%-60% of patients experience disease progression after 11-18 months of treatment, and its resistance mechanisms are complex and diverse: 15%-20% of patients lose the covalent binding site of the drug to the kinase domain due to the EGFR C797S mutation; 5%-10% of patients have MET gene amplification, activating the ERBB3-PI3K bypass signal; some other patients escape drug inhibition through mechanisms such as RAS-MAPK pathway activation, HER2 amplification, or histological transformation into small cell lung cancer.

[0003] Existing solutions focus on the research and development of fourth-generation TKIs and combination treatment strategies, but both have significant limitations. For example, although fourth-generation TKIs targeting the C797S / T790M / 19del triple mutation (such as EAI045 and BLU-945) showed activity in preclinical models, their poor selectivity and high off-target toxicity limit clinical translation; the combination of osimertinib with MET inhibitors (such as savolitinib) or MEK inhibitors (such as trametinib) is only effective for specific resistant subtypes (such as MET amplification), and the superimposed toxicity leads to an increased incidence of dose-limiting adverse reactions. In addition, there is currently a lack of a biomarker detection system for efficiently predicting resistance mechanisms, making it difficult to achieve dynamic and precise adjustment of treatment regimens. Summary of the Invention

[0004] In view of the deficiencies existing in the prior art, the present invention provides a pharmaceutical composition for sensitizing the treatment of EGFR-TKI-resistant tumors and its application. It solves the problem of NSCLC resistance to EGFR-TKI existing in the prior art.

[0005] In the first aspect of the present invention, there is provided a pharmaceutical composition, which comprises a) a therapeutically effective amount of decitabine and b) a therapeutically effective amount of an EGFR tyrosine kinase inhibitor.

[0006] In one embodiment of the present invention, the EGFR tyrosine kinase inhibitor comprises one or more of gefitinib, erlotinib, afatinib, and osimertinib.

[0007] In one embodiment of the present invention, the EGFR tyrosine kinase inhibitor is osimertinib.

[0008] In one embodiment of the present invention, the decitabine and the EGFR tyrosine kinase inhibitor are formulated into separate unit doses for simultaneous, separate, or sequential administration.

[0009] In one embodiment of the present invention, components such as decitabine and the EGFR tyrosine kinase inhibitor in the pharmaceutical composition can be administered separately at different times during the treatment process, or simultaneously in the form of separate unit doses. In one embodiment, the components in the pharmaceutical composition can be administered daily, once or twice a day, or weekly.

[0010] In one embodiment of the present invention, the effective doses of the components in the pharmaceutical composition can vary depending on the specific compound used, the mode of administration, the disease being treated, and the severity of the disease being treated. Therefore, the dose of the pharmaceutical composition of the present invention is selected according to various factors, including the route of administration and the renal and liver functions of the patient, etc.

[0011] In one embodiment of the present invention, the ratios, individual and combined doses, and concentrations of the components in the pharmaceutical composition are based on the kinetics of the components and can be determined using methods known to those skilled in the art. When the components in the pharmaceutical composition of the present invention are administered in the form of a single drug, their doses and administration methods can be based on the information provided in the package inserts of the corresponding commercially available drugs.

[0012] In one embodiment of the present invention, by mass ratio, decitabine:EGFR tyrosine kinase inhibitor = (30 - 100):(30 - 250).

[0013] In one embodiment of the present invention, the decitabine can be administered to a suitable subject daily in a single dose or divided doses, and the effective dose is about 0.05 to about 0.5 mg per kilogram of body weight per day. For example, about 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50 mg / kg / day.

[0014] In one embodiment of the present invention, the EGFR tyrosine kinase inhibitor can be administered to a suitable subject daily in a single dose or divided doses, and the effective dose is 30 - 250 mg / day. For example, about 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mg / day.

[0015] In one embodiment of the present invention, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0016] In one embodiment of the present invention, the carrier includes but is not limited to: a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, such as silica, talc, stearic acid, its magnesium or calcium salts and / or polyethylene glycol; c) binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; d) disintegrants, such as starch, agar, alginic acid or its sodium salt, or effervescent mixtures; e) absorbents, colorants, flavorants, sweeteners, preservatives, stabilizers, wetting agents or emulsifiers. Those of ordinary skill in the art can select one or more of the above carriers according to the properties specifically required for the dosage form through routine experiments. The amount of each carrier used can vary within the conventional range in the art.

[0017] In one embodiment of the present invention, the pharmaceutical composition further comprises other anti-tumor agents, cytotoxic agents, chemotherapeutic agents, cell inhibitors, immunomodulators or any combination thereof.

[0018] In one embodiment of the present invention, the pharmaceutical composition can be administered in vivo, in vitro or ex vivo. In one embodiment of the present invention, the pharmaceutical composition can be applied to host cells, especially human host cells. In one embodiment of the present invention, the pharmaceutical composition is administered by any conventional route, including enteral oral, parenteral, intravenous, intranasal, intratumoral and intramuscular administration routes.

[0019] In one embodiment of the present invention, when decitabine and an EGFR tyrosine kinase inhibitor are co-administered, they have a synergistic effect.

[0020] In one embodiment of the present invention, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect of inhibiting the proliferation of drug-resistant cells.

[0021] In one embodiment of the present invention, the effect of the pharmaceutical composition in inhibiting the proliferation of drug-resistant cells gradually increases with the increase in concentration.

[0022] In one embodiment of the present invention, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect of promoting apoptosis of drug-resistant cells.

[0023] In one embodiment of the present invention, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect of inhibiting tumor growth. In one embodiment of the present invention, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect of inhibiting tumor volume and / or tumor weight.

[0024] In one embodiment of the present invention, the pharmaceutical composition can activate the autophagy pathway.

[0025] In one embodiment of the present invention, the pharmaceutical composition can increase the LC3-II / LC3-I ratio, increase the total LC3-II level, decrease the p62 protein level, and increase the TP53INP1 protein expression level. Specifically, the pharmaceutical composition has the following characteristics: a) upregulating the LC3-II lipidation level to promote autophagosome formation; b) decreasing the p62 / SQSTM1 protein stability to enhance the clearance of ubiquitinated substrates; c) activating the autophagy-apoptosis signal synergistic pathway mediated by TP53INP1.

[0026] In one embodiment of the present invention, compared with the EGFR tyrosine kinase inhibitor alone, the pharmaceutical composition can more significantly promote the decrease in the methylation level of the TP53INP1 promoter.

[0027] In the second aspect of the present invention, there is provided the use of the above-mentioned pharmaceutical composition in the preparation of a drug for treating cancer.

[0028] In one embodiment of the present invention, the cancer is a BRAF-related cancer. In one embodiment of the present invention, the cancer is a cancer with a mutated BRAF-kinase. In one embodiment of the present invention, the cancer is a cancer resistant to EGFR-TKI. In one embodiment of the present invention, the cancer is non-small cell lung cancer.

[0029] In one embodiment of the present invention, the application includes at least one of the following:

[0030] 1) Improving tumor killing activity;

[0031] 2) Inhibiting tumor proliferation and growth;

[0032] 3) Promoting tumor cell apoptosis;

[0033] 4) Activating the autophagy pathway;

[0034] 5) Increasing the LC3-II / LC3-I ratio, increasing the total LC3-II level, decreasing the p62 protein level, and increasing the TP53INP1 protein expression level;

[0035] 6) Promoting a decrease in the methylation level of the TP53INP1 promoter.

[0036] In the third aspect of the present invention, the use of LC3, p62, and / or TP53INP1 as drug targets in screening drugs for treating EGFR-TKI-resistant cancers.

[0037] In one embodiment of the present invention, the drugs for treating EGFR-TKI-resistant cancers include LC3 expression promoters, p62 expression inhibitors, and TP53INP1 expression promoters.

[0038] In one embodiment of the present invention, the drugs for treating EGFR-TKI-resistant cancers have the following characteristics:

[0039] 1) Tumor killing activity;

[0040] 2) Inhibiting tumor proliferation and growth;

[0041] 3) Promoting tumor cell apoptosis;

[0042] 4) Activating the autophagy pathway;

[0043] 5) Increasing the LC3-II / LC3-I ratio, increasing the total LC3-II level, decreasing the p62 protein level, and increasing the TP53INP1 protein expression level;

[0044] 6) Promoting a decrease in the methylation level of the TP53INP1 promoter.

[0045] In the fourth aspect of the present invention, there is provided the use of decitabine in the preparation of a drug for enhancing the efficacy of EGFR-TKI-resistant tumors.

[0046] In the fifth aspect of the present invention, there is provided the use of decitabine in the preparation of a drug for enhancing the efficacy of an EGFR tyrosine kinase inhibitor.

[0047] In one embodiment of the present invention, the EGFR tyrosine kinase inhibitor is osimertinib.

[0048] In the sixth aspect of the present invention, a method for screening a substance that inhibits EGFR-TKI-resistant tumors or enhances the efficacy of EGFR-TKI-resistant tumors is provided. The method includes: (1) contacting a candidate substance with a system containing an autophagy flux signaling pathway; (2) screening out a substance that regulates the autophagy flux signaling pathway.

[0049] Wherein, the autophagy flux signaling pathway includes LC3-II protein, p62 protein and / or TP53INP protein, and the regulation includes promoting LC3 expression, promoting TP53INP1 expression, inhibiting p62 expression, and promoting autophagy flux.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] This combined treatment regimen directly targets EGFR-TKI-resistant tumors, especially the osimertinib resistance problem caused by EGFR mutations in non-small cell lung cancer (NSCLC). It has a clear therapeutic target, high clinical translation potential, provides new treatment options for patients, and provides a theoretical basis and practical reference for the formulation of future individualized treatment plans. As a DNA methyltransferase inhibitor, decitabine reverses drug resistance through epigenetic regulation, providing new ideas and methods for overcoming osimertinib resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a result diagram of CCK8 experiment verifying the proliferation inhibitory effect of decitabine on osimertinib-resistant cells. Among them, Figure 1 A is the inhibition result of different drug groups on PC-9OR; Figure 1 B is the inhibition result of different drug groups on H1975OR; Figure 1 C is the curve of the inhibitory effect of Deci on PC-9OR changing with concentration and the IC50 value of Deci in PC-9OR cells; Figure 1 D is the curve of the inhibitory effect of Deci on H1975OR changing with concentration and the IC50 value of Deci in H1975OR cells;

[0053] Figure 2It is the result diagram of flow cytometry experiment to verify the synergistic effect of decitabine and osimertinib in promoting apoptosis of drug-resistant cells. Among them, Figure 2 A is the result of PC-9OR cells, Figure 2 B is the result of H1975OR cells;

[0054] Figure 3 It is the result diagram of cell colony formation experiment to verify the inhibitory effect of decitabine and osimertinib in combination on the proliferation of drug-resistant cells. Among them, Figure 3 A is the result of PC-9OR cells, Figure 3 B is the result of H1975OR cells;

[0055] Figure 4 It is the result diagram of EDU experiment to verify the effect of the combination of decitabine and osimertinib on the proliferation of drug-resistant cells from the morphological perspective. Among them, Figure 4 A is the result of PC-9OR cells, Figure 4 B is the result of H1975OR cells;

[0056] Figure 5 It is the result diagram of animal experiment to verify the effect of decitabine combined with osimertinib in overcoming drug resistance. Among them, Figure 5 A is the experimental process diagram; 5B and 5C are the results of tumor volume changes in each group, Figure 5 D is the result of the change in tumor weight in each group;

[0057] Figure 6 It is the result diagram of the effect of decitabine combined with osimertinib on gene expression. Among them, Figure 6 A is PC-9OR cells, Figure 6 B is H1975OR cells;

[0058] Figure 7 It is the detection result of the methylation status of the TP53INP1 gene. 7A is the methylation ratio of TP53INP1, and 7B is the non-methylation ratio of TP53INP1; 7C and 7D are the methylation status of CpG sites;

[0059] Figure 8 It is the result of the expression of autophagy-related proteins in drug-resistant cells treated with decitabine combined with osimertinib. 8A is the protein imaging diagram, and 8B is the relative expression levels of p62 and LC-3II / I;

[0060] Figure 9 It is the detection result of autophagy activation in the reversal of osimertinib resistance by decitabine. Among them, Figure 9 A-D are the subcellular expression results of LC3 and TP53INP1, Figure 9 E-H are the results of the relative expression levels of TP53INP1, p62 and LC-3II / I. Detailed implementation methods

[0061] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0062] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0063] The terms "synergistic" and "synergism" mean that the effect obtained by using the compounds simultaneously is higher than the sum of the effects obtained by using these compounds alone, that is, higher than the effect that can be expected based on the separate administration of these two active ingredients. A synergistic effect can be obtained when the compounds are in the following situations: (1) formulated and administered or delivered in the form of a combined preparation; (2) delivered alternately or in parallel in the form of separate preparations; or (3) administered by some other protocol. When delivered in an alternating therapy, a synergistic effect can be obtained if the compounds are administered or delivered continuously, for example, in the form of separate tablets, pills or capsules, or different injections contained in separate syringes. In an alternating therapy, the effective doses of the various active ingredients are administered continuously, that is, in sequence; in a combined therapy, the effective doses of two or more active ingredients are administered simultaneously. A synergistic antitumor effect means an antitumor effect that is higher than the effect expected by simply adding up the effects of the individual compounds in the combination.

[0064] The term "carrier" is intended to include, if applicable, any solvent, dispersion medium, coating, diluent, buffer, isotonic agent, solution, suspension, colloid, inert substance, etc. or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for therapeutic or diagnostic purposes.

[0065] The term "effective amount" means an amount that is capable of treating or ameliorating a disease or disorder or capable of producing a desired therapeutic effect.

[0066] A "therapeutic agent" can be any physiologically or pharmacologically active substance that can produce a desired biological effect at a target site in a subject. The therapeutic agent can be a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleolytic compound, a radioactively active isotope, a receptor, and a prodrug-activating enzyme, which can be naturally occurring or produced by synthetic or recombinant methods or any combination thereof. Drugs affected by classical multi-drug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), anthracyclines (e.g., doxorubicin and daunomycin), RNA transcription inhibitors (e.g., actinomycin-D), and microtubule-stabilizing drugs (e.g., paclitaxel), may have special uses as therapeutic agents. Cytokines can also be used as therapeutic agents. Examples of these cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cancer chemotherapeutic agents may be preferred therapeutic agents. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art can refer to a large number of reference manuals, including but not limited to the Physician's Desk Reference and the 10th edition of Goodman and Gilman's Pharmacological Basis of Therapeutics, edited by Hardman et al., 2001.

[0067] "Pharmaceutically acceptable" means a molecular entity and composition that, when administered to a human, do not produce an allergic or similar untoward reaction.

[0068] Example 1 Inhibitory effect of decitabine on the proliferation of osimertinib-resistant cells

[0069] The osimertinib (Osimertinib, abbreviated as Osi)-resistant cells PC-9OR and H1975OR were seeded into 96-well plates at an appropriate density (usually 3000 - 5000 cells per well), and 100 μL of complete medium (DMEM or RPMI-1640 medium containing 10% fetal bovine serum) was added to each well. Prepare decitabine solutions at different concentrations (0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, etc.) (Decitabine, abbreviated as Deci), and dilute them to the required concentration with complete medium. Aspirate the medium in the 96-well plates, and add 100 μL of medium containing different concentrations of decitabine + 0.5 μM Osi to each well. Set 3 - 5 replicates for each concentration, and set up a blank control group (containing only medium, without cells) and a negative control group (containing cells, without drug treatment), as well as positive control group 1 with Osi alone (H1975 cells + Osi), positive control group 2 (H1975OR resistant cells + Osi), positive control group 3 (PC-9 cells + Osi), and positive control group 2 (PC-9OR resistant cells + Osi). Place the 96-well plates back into the incubator and continue culturing for 48 hours. At the predetermined time point, add 10 μL of CCK8 reagent to each well (pay attention to avoid generating bubbles). Gently shake the 96-well plates to evenly distribute the reagent, and then place the plates back into the incubator and continue incubating for 2 hours. Measure the absorbance (OD value) of each well at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. Record the OD value of each well and calculate the average value.

[0070] Calculate the cell proliferation inhibition rate:

[0071]

[0072] Use software such as GraphPad Prism to plot the cell proliferation inhibition curve, and calculate that the IC50 values of decitabine in PC-9OR and H1975OR cells are 1 μM and 1.2 μM respectively. The results are as Figure 1 shown.

[0073] It can be seen from the results that the inhibitory effect of the combined use of decitabine and osimertinib on the proliferation of resistant cells is significantly enhanced, and the proliferation inhibition effect gradually increases with the increase in the concentration of decitabine.

[0074] Example 2 Synergistic pro-apoptotic effect of the combined application of decitabine and osimertinib

[0075] Seed the osimertinib-resistant cells PC-9OR and H1975OR into 6-well plates at an appropriate density (1×10 per well , 5 ,

[0075] , ,

[0074] , ,

[0073] For each well containing 1×10⁶ cells, add 2 mL of RPMI-1640 medium containing 10% fetal bovine serum. Place the cells in an incubator at 37 °C and 5% CO₂ overnight to allow the cells to adhere and enter the logarithmic growth phase. Divide the experiment into four groups: control group, decitabine monotherapy group, osimertinib monotherapy group, and combination treatment group of decitabine and osimertinib (the added amounts are 0.5 μM Osi and 5 μM Deci). After treating the cells with the drugs, continue culturing for 48 hours. Digest the cells with trypsin and collect the cell suspension into a centrifuge tube. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash the cells twice with pre-cooled PBS. Operate according to the Annexin V-FITC / PI apoptosis detection kit instructions: Resuspend the cells in 100 μL of Annexin V binding buffer, add 5 μL of Annexin V-FITC and 5 μL of PI (propidium iodide), mix gently, and incubate in the dark at room temperature for 15 minutes. After the incubation, immediately add 400 μL of Annexin V binding buffer, mix well, and use a flow cytometer to detect the apoptosis rate of the cells. Calculate the apoptosis rate of each group of cells (early apoptosis rate + late apoptosis rate). Compare the apoptosis rates of the control group, monotherapy groups, and combination treatment group to verify the synergistic effect of decitabine and osimertinib. The results are as Figure 2 shown.

[0076] As can be seen from the results, the apoptosis rate of the combination treatment group of decitabine and osimertinib on drug-resistant cells was significantly higher than that of the decitabine monotherapy group and the osimertinib monotherapy group, indicating that decitabine and osimertinib have a synergistic effect in promoting the apoptosis of drug-resistant cells.

[0077] Example 3 Inhibitory effect of the combination of decitabine and osimertinib on the proliferation of drug-resistant cells

[0078] The osimertinib-resistant cells PC-9O and H1975O were seeded at a low density into 6-well plates (200 - 1000 cells per well). 2 mL of complete medium was added to each well to ensure uniform distribution of the cells. The 6-well plates were placed in an incubator at 37 °C and 5% CO2 for the cells to adhere and start proliferating. The experiment was divided into four groups: a control group, a decitabine monotherapy group, an osimertinib monotherapy group, and a combination therapy group of decitabine and osimertinib (added amounts: 0.5 μM Osi and 2.5 μM Deci). 24 hours after cell seeding, the medium was aspirated, and fresh medium containing the corresponding drugs was added. Three replicate wells were set up for each group. The 6-well plates were placed back in the incubator for continued culture, and the fresh medium containing the drugs was changed every 2 - 3 days. Continuous culture was carried out for 10 - 14 days (the specific time was adjusted according to the cell proliferation rate) until visible cell clones were formed in the control group (usually colonies containing more than 50 cells). During the culture process, the cell growth status was observed regularly to avoid over-confluence of the cells. The medium was aspirated, and the cells were gently washed twice with pre-cooled PBS to remove residual medium and drugs. 1 mL of 4% paraformaldehyde solution was added to each well and fixed at room temperature for 15 minutes. The fixing solution was aspirated, and the cells were washed once with PBS. 1 mL of 0.1% crystal violet staining solution (prepared with deionized water) was added to each well and stained at room temperature for 15 - 30 minutes. The staining solution was aspirated, and the well plates were gently washed with deionized water to remove excess dye until the background was clean. The 6-well plates were air-dried, and the number of clones formed in each well was observed and counted using the naked eye or a microscope. Image analysis software such as ImageJ was used to quantitatively analyze the clones, and the colony formation rate was calculated:

[0079]

[0080] The colony formation rates of the control group, the monotherapy groups, and the combination therapy group were compared to verify the inhibitory effect of the combination of decitabine and osimertinib on cell proliferation. The experimental results are as Figure 3 shown.

[0081] As can be seen from the results, the colony formation rate of the resistant cells in the combination therapy group of decitabine and osimertinib was significantly lower than that of the decitabine monotherapy group and the osimertinib monotherapy group, indicating that decitabine and osimertinib have a synergistic effect in inhibiting the proliferation of resistant cells.

[0082] Example 4 Effect of the combination of decitabine and osimertinib on the proliferation of resistant cells

[0083] The osimertinib-resistant cells PC-9OR and H1975OR were seeded at an appropriate density into 24-well plates or cell culture slides (5×10 4cells), and 1 mL of complete culture medium was added to each well. The cells were cultured in an incubator at 37°C and 5% CO2 overnight to allow the cells to attach and enter the logarithmic growth phase. The experiment was divided into four groups: control group, decitabine monotherapy group, osimertinib monotherapy group, and combination drug group (added amount 0.5 μM Deci). The drug-treated cells were cultured for a further 48 hours. Aspirate the culture medium and gently wash the cells twice with pre-cooled PBS to remove residual culture medium and drugs. Add 1 mL of 4% paraformaldehyde solution to each well and fix at room temperature for 15 minutes. Aspirate the fixative and wash once with PBS. Add 1 mL of 0.5% Triton X-100 (prepared with PBS) to each well and permeabilize at room temperature for 10 minutes to increase the permeability of the cell membrane. Aspirate the permeabilization solution and wash once with PBS. According to the EDU detection kit (such as BeyoClick TM Prepare Click reaction solution (containing fluorescent dye, buffer and catalyst) according to the instructions of EdU-555). Add 500μL Click reaction solution to each well and incubate at room temperature in the dark for 30 minutes. Aspirate the reaction solution and wash twice with PBS to remove unbound dye. Add 1mL Hoechst nuclear staining solution (1μg / mL, prepared with PBS) to each well and incubate at room temperature in the dark for 10 minutes to stain the cell nuclei. Aspirate the staining solution and wash twice with PBS. Place the cell slide or 24-well plate under a fluorescence microscope for observation. Use appropriate excitation and emission light wavelengths to capture images of EDU-positive cells and cell nuclei, respectively. Randomly select 5-10 fields of view for image acquisition in each group to ensure the representativeness of the data.

[0084] Use image analysis software such as ImageJ to count the EdU-positive cells and calculate the EdU-positive cell rate:

[0085]

[0086] The EDU-positive cell rates of the control group, single-drug group, and combination group were compared to verify the inhibitory effect of decitabine and osimertinib combination on the proliferation of drug-resistant cells. Figure 4 shown.

[0087] The results showed that the inhibitory effect of the decitabine and osimertinib combination group on drug-resistant proliferation was significantly higher than that of the decitabine monotherapy group and the osimertinib monotherapy group, indicating that decitabine and osimertinib have a synergistic effect in inhibiting the proliferation of drug-resistant cells.

[0088] Example 5 Animal experiment on overcoming drug resistance by combining decitabine with osimertinib

[0089] Select healthy nude mice (BALB / c nude mice) at 6-8 weeks of age, ensuring that the mice have similar body weights (usually 18-22 g). House the mice in an SPF-class animal room, maintaining a constant temperature (22-25 °C), constant humidity (50-60%), and a 12-hour light / dark cycle, and providing sufficient food and water. Culture osimertinib-resistant cells H1975OR and PC-9OR, collect cells in the logarithmic growth phase, resuspend them with PBS, and adjust the cell density to 1×10 7 cells / mL. Inoculate the cell suspension subcutaneously into the right dorsal side of the nude mice, with 100 μL (containing 1×10 6 cells) inoculated into each mouse. Observe the status of the mice daily. When the tumor volume reaches approximately 50-100 mm 3 , start the grouped experiment. Randomly divide the tumor-bearing mice into four groups (5 mice in each group): Control group: Administer PBS or normal saline with the same volume as the drug solvent; Osimertinib monotherapy group: Administer osimertinib by intraperitoneal injection at 5-10 mg / kg; Decitabine monotherapy group: Administer decitabine by intraperitoneal injection at 1-5 mg / kg; Combination therapy group: Administer osimertinib and decitabine simultaneously (at the same doses as the monotherapy groups). The treatment starts on the day of grouping and lasts for 4 weeks. Record the body weight and tumor volume of the mice daily. Measure the long diameter (L) and short diameter (W) of the tumor using a vernier caliper every 2-3 days, and calculate the tumor volume according to the following formula:

[0090]

[0091] Record the tumor volume measured each time, and present the data in the form of mean±SEM (n = 5).

[0092] At the end of the treatment (4 weeks), sacrifice the mice, remove the tumor tissues and weigh them. Record the average weight of the tumors in each group and perform statistical analysis. At the same time, collect tumor tissue samples: Immediately freeze part of the tumor tissue at -80 °C for subsequent Western blotting analysis. Fix part of the tumor tissue in 4% paraformaldehyde for HE staining or immunohistochemistry. Compare the differences in tumor volume and weight among the groups, and use t-test or ANOVA to analyze the significance (*P<0.05; **P<0.01; ***P<0.001). Calculate the tumor growth inhibition rate:

[0093]

[0094] Show the changes in tumor volume and weight in each group. Mark the significant differences (*P<0.05; **P<0.01; ***P<0.001) in the chart. The results are as Figure 5 shown.

[0095] The results show that the inhibitory effect of the combination of decitabine and osimertinib on the growth of drug-resistant tumors is significantly higher than that of the decitabine monotherapy group and the osimertinib monotherapy group, indicating that decitabine and osimertinib have a synergistic effect on inhibiting the growth of drug-resistant tumors.

[0096] Example 6 Effects of Decitabine Combined with Osimertinib on the Expression of Different Genes in Drug-Resistant Cell Lines

[0097] After trypsinizing PC-9OR and H1975OR cells in the logarithmic growth phase, adjust the cell density and seed them into 96-well plates and 12-well plates respectively. In the 96-well plates, seed 5000 - 10000 cells per well, and in the 12-well plates, seed 50000 - 100000 cells per well. Add an appropriate amount of medium, gently shake the culture plates to evenly distribute the cells, and incubate them in the incubator for 24 h until the cells adhere to the wall. Conduct the following groupings respectively: PC-9OR / H1975OR control group, Osi treatment group, Deci treatment group, Osi + Deci combination treatment group. Add solutions of Osi with a final concentration of 1 μM and Deci with a final concentration of 5 μM to the wells simultaneously. Set at least 3 replicate wells for each treatment group. Place the treated cell culture plates back into the incubator and continue culturing for 48 h. After the culture is completed, discard the medium in the 12-well plates, gently rinse the cells 2 - 3 times with pre-cooled PBS buffer to remove the residual medium. Add 1 ml of TRIzol reagent to each well, let it stand at room temperature for 5 min to allow TRIzol to fully lyse the cells. Use a pipette to repeatedly pipette the cell lysate to ensure complete cell lysis with no obvious cell clumps. Transfer the lysate to a RNase-free centrifuge tube, add 200 μl of chloroform, vigorously shake for 15 s, and let it stand at room temperature for 2 - 3 min. Centrifuge at 4°C and 12000 g for 15 min. At this time, the solution is divided into three layers: the upper layer is a colorless aqueous phase (containing RNA), the middle layer is a white protein layer, and the lower layer is a red organic phase. Carefully aspirate the upper aqueous phase and transfer it to a new centrifuge tube, avoiding aspirating the middle and lower layer liquids. Add an equal volume of isopropanol to the aqueous phase, gently invert and mix well, and let it stand at room temperature for 10 min to precipitate the RNA. Centrifuge at 4°C and 12000 g for 10 min, discard the supernatant, and a white RNA precipitate can be seen at the bottom of the centrifuge tube. Wash the RNA precipitate twice with 75% ethanol (prepared with DEPC water), add 1 ml of ethanol each time, gently invert the centrifuge tube, centrifuge at 4°C and 7500 g for 5 min, and discard the supernatant. Air-dry the RNA precipitate at room temperature for 5 - 10 min until there is no obvious ethanol residue on the surface of the RNA precipitate, add an appropriate amount of DEPC water to dissolve the RNA, and incubate at 55 - 60°C for 10 - 15 min to promote RNA dissolution. Use a nucleic acid protein analyzer to measure the RNA concentration and purity, ensure that the A260 / A280 ratio is between 1.8 - 2.0 and the A260 / A230 ratio is greater than 2.0, and store the RNA samples in a -80°C refrigerator for later use. According to the reverse transcription kit instructions, prepare the reverse transcription reaction system. Sequentially add 500 ng - 1 μg of total RNA, 1 μl of random primer or Oligo(dT) primer, and 1 μl of 10 mM dNTP Mix into a 0.2 ml RNase-free PCR tube, and make up to 12 μl with DEPC water, gently mix well. Place the reaction tube in a PCR instrument, incubate at 65°C for 5 min, and quickly place it on ice to cool for 2 - 3 min.Add 4 μl of 5× reverse transcription buffer, 2 μl of 0.1 M DTT, 1 μl of RNase inhibitor, and 1 μl of reverse transcriptase to the reaction tube, and gently mix to make the total volume reach 20 μl. Place the reaction tube in a PCR instrument and perform reverse transcription reaction according to the program of incubating at 37°C for 60 min and 70°C for 15 min. After the reaction, store the cDNA product in a -20°C refrigerator for later use. Design and synthesize specific primers according to the sequences of the target genes (such as TP53INP1, SLC5A10, etc.) and reference genes (such as β-actin, GAPDH). The primer sequences need to be designed by primer design software and verified by BLAST alignment to ensure primer specificity.

[0098] Prepare the real-time fluorescence quantitative PCR reaction system. In a 96-well optical reaction plate, add 10 μl of 2× SYBR Green PCR Master Mix, 0.5 μl of each upstream and downstream primer (final concentration is 0.2 - 0.5 μM), and 2 μl of cDNA template to each well, and make up to 20 μl with sterile deionized water. Gently mix, cover the optical reaction plate lid, and centrifuge at 300 - 500 g for 1 - 2 min to fully mix the reaction solution and remove air bubbles. Place the reaction plate in a real-time fluorescence quantitative PCR instrument and perform amplification reaction according to the program recommended by the instrument. Generally, the reaction conditions are: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, for a total of 40 cycles; after the reaction, perform melting curve analysis to verify the specificity of the amplification product.

[0099] Set 3 technical replicates for each sample, and calculate the fold change in the expression of the target gene using the relative quantification method (2-ΔΔCt method). Using the gene expression level of the control group as a reference, calculate the fold change in the expression of the target gene in each treatment group relative to the control group. The formula is: fold change = 2-ΔΔCt, where ΔΔCt = (Ct target gene - Ct reference gene)treatment group - (Ct target gene - Ct reference gene)control group. Use data analysis software such as GraphPad Prism to perform statistical analysis on the results of real-time fluorescence quantitative PCR. Use one-way ANOVA or other appropriate statistical methods to compare the significance of differences in the fold change of gene expression between different treatment groups. A P < 0.05 is considered to indicate a statistically significant difference. The results are as Figure 6 shown.

[0100] The results show that when PC-9OR and H1975OR drug-resistant cells are treated with osimertinib alone, decitabine alone, or the combination of the two drugs, there are significant differences in the gene expression of TP53INP1, SLC5A10, SYTL1, GABBR2, and JAK2, and the differences are statistically significant. For the two cell lines, PC-9OR and H1975OR, compared with other genes with differential expression, in the group treated with osimertinib alone, the gene expression level of TP53INP1 is lower than that of the control group; in the group treated with decitabine alone, the gene expression level of TP53INP1 is significantly increased; when the two drugs are used in combination, the expression level of TP53INP1 is between that of the groups treated with osimertinib and decitabine alone, and the difference is statistically significant, suggesting that decitabine may restore the expression of TP53INP1 in drug-resistant cells.

[0101] Example 7 Effect of Decitabine on the Methylation Status of TP53INP1 Gene in PC-9OR and H1975OR Drug-Resistant Cell Lines

[0102] PC-9OR and H1975OR cells were each divided into a control group and different treatment groups, with 3 biological replicates in each group. The corresponding treatment factors, such as specific drugs, stimulants, etc., were applied to the cells in each treatment group, while the control group was not treated or only equal amounts of solvent were added. After treatment for a certain period of time (determine the appropriate duration according to pre-experiments or literature, such as 24 hours, 48 hours, etc.), the cells were collected for subsequent analysis. The genomic DNA of PC-9OR and H1975OR cells in each experimental group and the control group was extracted using a DNA extraction kit according to the instructions. The concentration and purity of the DNA were detected using a spectrophotometer to ensure A 260 / A 280The ratio is between 1.8 and 2.0, and it is stored for standby at -20°C. The methylation level of the TP53INP1 gene is detected by methylation-specific PCR (MSP) or bisulfite sequencing (BSP). The extracted genomic DNA is modified with bisulfite to convert unmethylated cytosine into uracil, while methylated cytosine remains unchanged. Methylation- and non-methylation-specific primers are designed according to the modified sequence for PCR amplification. The amplified bands are observed by agarose gel electrophoresis to judge the methylation status of the gene, and then the change in methylation level is analyzed. The bisulfite-modified DNA is subjected to PCR amplification, and the amplified product is cloned into a vector. Positive clones are selected for sequencing. The sequencing results are analyzed to calculate the methylation level of the gene, that is, the proportion of methylated cytosine to total cytosine. The bisulfite sequencing method (BSP) or methylated DNA immunoprecipitation sequencing (MeDIP-seq) is used to determine the methylation status of specific CpG sites of the TP53INP1 gene. PCR amplification, cloning, and sequencing are performed on specific regions, and the sequencing results are analyzed to visually present methylation (black) and non-methylation (white) in the form of black and white circles, and the methylation ratio of each CpG site is statistically analyzed. Methylated DNA fragments are enriched with 5-methylcytosine antibody, a sequencing library is constructed, and high-throughput sequencing is performed. Through bioinformatics analysis, the methylation status of specific CpG sites is determined, and a methylation map is drawn. The results are as Figure 7 shown.

[0103] It can be seen from the results that decitabine reverses the decreased expression of TP53INP1 in PC-9OR and H1975OR drug-resistant cell lines through demethylation modification of TP53INP1.

[0104] Example 8 Effect of Decitabine Combined with Osimertinib on the Expression of Autophagy-Related Proteins in Drug-Resistant Cells

[0105] PC-9OR and H1975OR cells were grouped for experiments. For each cell line, two groups were set up. One group was treated only with Osi (Osimertinib), labeled as the Osi group; the other group was treated with a combination of Osi and Deci (Decitabine), labeled as the Osi+Deci group. Each group was set with 3 replicate wells. According to the concentrations designed in the experiment, Osi or a mixture of Osi and Deci was added to the corresponding cell culture system, and the control group was added with an equal amount of drug-free culture medium. Incubate in the incubator for a certain period of time (determined according to preliminary experiments or references, such as 48 hours). After the treatment time ended, discard the medium and wash the cells 2-3 times with pre-cooled PBS. Add an appropriate amount of RIPA lysis buffer containing protease inhibitor to each well, lyse on ice for 30 minutes, collect the cell lysate, centrifuge at 4°C and 12,000 rpm for 15 minutes, and take the supernatant to obtain the total protein extract. The protein concentration was measured using a BCA protein quantification kit. A standard curve was made with bovine serum albumin (BSA), and the protein concentration of the sample was calculated according to the standard curve, and the protein concentrations of each sample were adjusted to be consistent. Prepare separating gel and stacking gel with appropriate concentrations (such as 12% separating gel and 5% stacking gel). Mix the protein sample with the loading buffer and boil for 5 minutes to denature the protein. Take an equal amount of protein sample and add it to the loading well, and at the same time add protein Marker, and perform SDS-PAGE electrophoresis to separate proteins with different molecular weights. After electrophoresis, electrotransfer the proteins on the gel to a PVDF membrane, and the transfer conditions can be set to a constant current of 250 mA for about 90 minutes. Place the PVDF membrane in TBST buffer containing 5% skim milk and block at room temperature for 1-2 hours to reduce non-specific binding. After blocking, incubate the membrane with primary antibodies against p62 and LC-3B overnight at 4°C. The next day, wash the membrane 3 times with TBST buffer, 10 minutes each time. Incubate the membrane with HRP-labeled secondary antibody (diluted according to the instruction manual) at room temperature for 1-2 hours. Wash the membrane 3 times again with TBST buffer, 10 minutes each time. Add ECL chemiluminescent substrate, expose and develop under a chemiluminescent imaging system to obtain protein band images. Use image analysis software to analyze the gray scale of the bands and calculate the relative expression levels of p62 and LC-3II / I. The results are as Figure 8 shown.

[0106] As can be seen from the results, compared with the Osi single-drug group, the combination of osimertinib and decitabine reduced the expression of p62 protein and increased the expression of LC-3II protein in drug-resistant cells, suggesting that the combination of osimertinib and decitabine may activate autophagy.

[0107] Example 9 Detection of autophagy activation in the reversal of osimertinib resistance by decitabine

[0108] Use NSCLC cell lines resistant to osimertinib (such as H1975 OR or PC-9OR) as experimental subjects. Set up experimental groups: control group, decitabine treatment group, autophagy activation group (rapamycin), and autophagy inhibition group (3-MA). The treatment concentration of osimertinib was determined as IC50 concentration / 2, 0.5 μM according to preliminary experiments; decitabine: the lowest dose was determined as 5 μM according to preliminary experiments; the drug concentration of rapamycin was 100 nM; the drug concentration of 3-MA was 5 mM. The cells were cultured in a 37 °C, 5% CO2 incubator for 48 hours. Aspirate the culture medium and wash the cells 3 times with pre-cooled PBS. Add 4% paraformaldehyde to fix the cells and place them at room temperature for 15 minutes. Aspirate the fixative and wash 3 times with PBS. Add 0.1% Triton X-100 (prepared with PBS) to permeabilize the cell membrane and place it at room temperature for 10 minutes. Wash 3 times with PBS. Add 5% BSA (bovine serum albumin) blocking solution and block at room temperature for 30 minutes. Aspirate the blocking solution and add primary antibodies (LC3 antibody and p62 antibody) respectively, and incubate overnight at 4 °C. Aspirate the primary antibody and wash 3 times with PBS, 5 minutes each time. Add fluorescently labeled secondary antibodies (such as Alexa Fluor 488-labeled anti-rabbit IgG and Alexa Fluor 594-labeled anti-mouse IgG), and incubate in the dark at room temperature for 1 hour. Aspirate the secondary antibody and wash 3 times with PBS, 5 minutes each time. Add DAPI staining solution (1 μg / mL) to stain the cell nuclei and incubate in the dark at room temperature for 5 minutes. Wash 3 times with PBS. Place the cells under a fluorescence microscope and use appropriate filter sets to detect LC3 (green fluorescence), p62 (red fluorescence), and DAPI (blue fluorescence) respectively. Randomly select 5 fields of view and take fluorescence images. Use ImageJ software to analyze the fluorescence intensity and quantify the expression levels of LC3 and TP53INP1. The results are as Figure 9 shown in A-D. Compared with the single-drug group, the subcellular expression levels of LC and TP53INP1 in the combination drug group were significantly increased.

[0109] H1975 OR and PC-9 OR were also used as experimental subjects. The experimental groups were set as follows: control group, decitabine treatment group, autophagy activation group (rapamycin), and autophagy inhibition group (3-MA). The treatment concentration of osimertinib was 0.5 μM; the treatment concentration of decitabine was 5 μM; the drug use concentration of rapamycin was 100 nM; the drug use concentration of 3-MA was 5 mM. Incubate in the incubator for a certain period of time (such as 48 hours determined according to the preliminary experiment). After the treatment time ended, discard the culture medium and wash the cells 2-3 times with pre-cooled PBS. Add an appropriate amount of RIPA lysis buffer containing protease inhibitor to each well and lyse on ice for 30 minutes. Collect the cell lysate, centrifuge at 12,000 rpm at 4°C for 15 minutes, and take the supernatant to obtain the total protein extract. The protein concentration was measured using a BCA protein quantification kit. A standard curve was made with bovine serum albumin (BSA), and the protein concentration of the sample was calculated according to the standard curve, and the protein concentrations of each sample were adjusted to be consistent. Prepare separation gel and stacking gel with appropriate concentrations (such as 12% separation gel and 5% stacking gel). Mix the protein sample with the loading buffer and boil for 5 minutes to denature the protein. Take an equal amount of protein sample and add it to the loading well, and at the same time add protein Marker for SDS-PAGE electrophoresis to separate proteins with different molecular weights. After electrophoresis, electrotransfer the proteins on the gel to the PVDF membrane, and the transfer conditions can be set to a constant current of 250 mA and the transfer time is about 90 minutes. Put the PVDF membrane into the TBST buffer containing 5% skim milk and block at room temperature for 1-2 hours to reduce non-specific binding. After blocking, incubate the membrane with primary antibodies against TP53INP1, p62, LC-3, and β-actin (diluted according to the instructions) overnight at 4°C. The next day, wash the membrane 3 times with TBST buffer, 10 minutes each time. Incubate the membrane with the HRP-labeled secondary antibody (diluted according to the instructions) at room temperature for 1-2 hours. Wash the membrane 3 times again with TBST buffer, 10 minutes each time. Add the ECL chemiluminescent substrate, expose and develop under the chemiluminescent imaging system to obtain the protein band image. Use image analysis software (such as Image J) to analyze the gray scale of the bands, use β-actin as the internal reference, calculate the relative expression levels of TP53INP1, p62, and LC-3II / I, and compare and analyze the data of different treatment groups. The results are as Figure 9 shown in E-H. When PC-9 OR and H1975 OR were treated with the combination of rapamycin and decitabine, the expression of p62 decreased to varying degrees in both groups of cells; the protein expressions of LC-3II and TP53INP1 showed an increasing trend, suggesting that the combined effect of decitabine and rapamycin may promote autophagy; when PC-9 OR and H1975 OR were treated with the combination of 3-MA and decitabine, compared with the treatment with decitabine alone, the expression of p62 decreased slightly; the expression of LC-3II increased slightly, indicating that the combined effect of decitabine and 3-MA may inhibit autophagy.

[0110] As can be seen from the results of the review, autophagy activation is an important factor in the reversal of osimertinib resistance by decitabine.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a) a therapeutically effective amount of decitabine and b) a therapeutically effective amount of an EGFR tyrosine kinase inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that: The EGFR tyrosine kinase inhibitor comprises one or more of gefitinib, erlotinib, afatinib, osimertinib; Preferably, the EGFR tyrosine kinase inhibitor is osimertinib.

3. The pharmaceutical composition according to claim 1, characterized in that: The decitabine and the EGFR tyrosine kinase inhibitor are formulated into separate unit doses for simultaneous, separate or sequential administration; Preferably, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other anti-tumor agents, cytotoxic agents, chemotherapeutic agents, cell inhibitors, immunomodulators or any combination thereof.

4. A pharmaceutical composition according to claim 1, characterized in that: When the decitabine and the EGFR tyrosine kinase inhibitor are co-administered, they have a synergistic effect.

5. A pharmaceutical composition according to claim 1, characterized in that: Compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect on inhibiting the proliferation of drug-resistant cells; Preferably, the effect of the pharmaceutical composition on inhibiting the proliferation of drug-resistant cells gradually increases with the increase of the concentration; Preferably, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect on promoting the apoptosis of drug-resistant cells; Preferably, compared with the EGFR tyrosine kinase inhibitor alone or decitabine alone, the pharmaceutical composition has a better effect on inhibiting tumor growth; Preferably, the pharmaceutical composition can activate the autophagy pathway; Preferably, the pharmaceutical composition can increase the LC3-II / LC3-I ratio, increase the total LC3-II level, decrease the p62 protein level, and increase the TP53INP1 protein expression level; Preferably, compared with the EGFR tyrosine kinase inhibitor alone, the pharmaceutical composition can more significantly promote the decrease of the methylation level of the TP53INP1 promoter.

6. Use of the pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament for treating cancer; Preferably, the cancer is BRAF-related cancer; preferably the cancer is a cancer with a mutated BRAF-kinase; preferably the cancer is a cancer resistant to EGFR-TKI; preferably the cancer is non-small cell lung cancer; Preferably, the use comprises at least one of the following: 1) Enhancing tumor killing activity; 2) Inhibiting the proliferation and growth of tumors; 3) Promoting apoptosis of tumor cells; 4) Activating the autophagy pathway; 5) Increasing the LC3-II / LC3-I ratio, increasing the total LC3-II level, decreasing the p62 protein level, and increasing the TP53INP1 protein expression level; 6) Promoting the decrease of the methylation level of the TP53INP1 promoter.

7. Use of LC3, p62 and / or TP53INP1 as a drug target in the screening of a medicament for treating EGFR-TKI-resistant cancer; Preferably, the medicament for treating EGFR-TKI-resistant cancer comprises an LC3 expression promoter, a p62 expression inhibitor, and a TP53INP1 expression promoter.

8. Use of decitabine in the preparation of a medicament for enhancing the efficacy of EGFR-TKI-resistant tumors.

9. Use of decitabine in the preparation of a drug for sensitizing the efficacy of an EGFR tyrosine kinase inhibitor; Preferably, the EGFR tyrosine kinase inhibitor is osimertinib.

10. A method for screening substances that inhibit EGFR-TKI-resistant tumors or enhance the efficacy of EGFR-TKI-resistant tumors, characterized in that: The method includes: (1) contacting a candidate substance with a system containing an autophagic flux signaling pathway; (2) screening out substances that regulate the autophagic flux signaling pathway; Wherein, the autophagic flux signaling pathway includes LC3-II protein, p62 protein, and / or TP53INP protein, and the regulation includes promoting LC3 expression, promoting TP53INP1 expression, inhibiting p62 expression, and promoting autophagic flux.

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