Application of phenazitinib in reversing osimertinib drug resistance

Abstract

The invention provides application of phenazitinib in reversing osimertinib drug resistance, and relates to the technical field of medicines. The research of the inventor finds that the phenanzotinib has the activity of resisting the acquired drug resistance of the lung cancer osimertinib, the phenanzotinib can target PIK3CA so as to regulate and control a PI3K / AKT pathway to induce autophagy, and by targeting DUT, cell G1 phase retardation and mitochondrial DNA damage are induced, and the activity of reversing the drug resistance of the lung cancer osimertinib is exerted; it is found that the phenanzantinib can be used as a PIK3CA and DUT double-target inhibitor, a candidate drug is provided for reversing osimertinib drug resistance of lung cancer, and experimental and theoretical bases are provided for clinical application of the anti-targeted drug resistance activity of the phenanzantinib.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a use of feizhuotinib in reversing osimertinib resistance. Background Art

[0002] Lung cancer is one of the most deadly cancers worldwide. Among patients with non-small cell lung cancer (NSCLC), epidermal growth factor receptor (EGFR) mutations are among the most common oncogenic driver mutations. Osimertinib, an irreversible, third-generation EGFR-TKI, selectively inhibits EGFR-sensitive mutations (exon 19 deletions and L858R mutations) and the T790M resistance mutation, while also exhibiting excellent central nervous system (CNS) activity. Osimertinib's FDA-approved indications include first-line treatment for patients with locally advanced or metastatic EGFRm non-small cell lung cancer (NSCLC), second-line treatment for patients with locally advanced or metastatic EGFR-T790M mutation-positive NSCLC, and adjuvant treatment for patients with early-stage EGFRm NSCLC. In addition, the drug has been approved in the United States and other countries for combination therapy with chemotherapy as a first-line treatment for patients with locally advanced or metastatic EGFRm NSCLC. Although osimertinib has demonstrated efficacy, similar to first- and second-generation EGFR-TKIs, tumor resistance is inevitable. Therefore, exploring treatments that can reverse osimertinib resistance could slow disease progression and is of great clinical significance.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The first object of the present invention is to provide the use of Fidotinib in the preparation of PIK3CA protein inhibitors.

[0005] The second object of the present invention is to provide the use of Fidotinib in the preparation of DUT protein inhibitors.

[0006] The third object of the present invention is to provide the use of feizhuotinib in the preparation of a reversal drug for reversing acquired resistance to osimertinib in lung cancer, so as to solve the above technical problems.

[0007] The fourth object of the present invention is to provide a use of fuzetinib combined with osimertinib in the preparation of a medicament for treating lung cancer.

[0008] The fifth object of the present invention is to provide a medicine for treating lung cancer.

[0009] In order to achieve the above objectives, the following technical solutions are adopted: In a first aspect, the present invention provides the use of feizotinib in the preparation of a PIK3CA protein inhibitor.

[0010] In a second aspect, the present invention provides the use of feizhuotinib in the preparation of DUT protein inhibitors.

[0011] In a third aspect, the present invention provides the use of feizhuotinib in the preparation of a reversal drug for reversing acquired resistance to osimertinib in lung cancer.

[0012] In a fourth aspect, the present invention provides the use of fuzetinib combined with osimertinib in the preparation of a medicament for treating lung cancer.

[0013] As a further technical solution, the lung cancer includes lung cancer with acquired resistance to osimertinib.

[0014] In a fifth aspect, the present invention provides a drug for treating lung cancer, comprising fuzetinib and osimertinib.

[0015] As a further technical solution, the drug also includes pharmaceutical excipients.

[0016] As a further technical solution, the pharmaceutical excipients include at least one of a filler, a binder, a disintegrant, a lubricant, a flavoring agent or a preservative.

[0017] As a further technical solution, the dosage form of the drug includes oral dosage form.

[0018] As a further technical solution, the oral dosage form is selected from tablets, capsules, granules and pills.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The inventors have discovered that Feizhuotinib has activity against acquired resistance to osimertinib in lung cancer. Feizhuotinib can target PIK3CA and then regulate the PI3K / AKT pathway to induce autophagy. By targeting DUT, it induces G1 phase arrest and mitochondrial DNA damage in cells, thereby reversing the resistance to osimertinib in lung cancer. The inventors found that Feizhuotinib can serve as a dual-target inhibitor of PIK3CA and DUT, providing a candidate drug for reversing osimertinib resistance in lung cancer, and providing experimental and theoretical basis for the clinical application of Feizhuotinib's anti-target resistance activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Figure 3: Osimertinib-resistant cell lines; (A) Schematic diagram of the construction of the osimertinib-resistant H1975 / OR cell line; (B) Microscopic observation of the morphological differences between H1975 and osimertinib-resistant H1975 / OR cells; (C) Cells were incubated with different concentrations of osimertinib for 48 h, and the IC50 values ​​of osimertinib against H1975 and H1975 / OR were determined using the CCK-8 assay; (D) Flow cytometry was used to detect cell apoptosis. Figure 2 : To observe the effect of combined treatment with feizotinib and osimertinib on the proliferation of drug-resistant cells; H1975 / OR cells were treated with osimertinib, feizotinib, or the combination of osimertinib and feizotinib, and then subjected to CCK-8 (A), EdU (B), colony formation (C), and wound healing assays (D); B. Scale bar: 200 μM; D. Scale bar: 100 μM; Figure 3 : Felotinib-induced cell cycle arrest in drug-resistant cells; (A) Flow cytometry analysis of cell cycle; (B) Western blotting to detect the expression of cell cycle-related proteins; **** p <0.0001; Figure 4 : Detection of apoptosis of drug-resistant cells induced by ferritinib; (A) Flow cytometry detection of cell apoptosis; (B) Western blotting detection of apoptosis-related protein expression; Figure 5 : Felotinib activates autophagy in H1975 / OR cells; (A) Western blotting to detect the expression of autophagy-related proteins in cells 24 h after drug treatment; (B) Fluorescence intensity of LC3 in cells after drug treatment; Scale bar: 200 μM; Figure 6: Representative images of fluorescent LC3 puncta; cell nuclei were stained with Hoechst; scale bar: 100 μM; Figure 7 Inhibition of autophagy weakens the anti-drug resistance activity of the combination therapy; (A) Flow cytometry was used to detect cell apoptosis after autophagy inhibition; (B) H1975 / OR cells were pretreated with CQ for 24 h and then treated with drugs, and cell viability was measured using the CCK-8 assay. Figure 8 : Felotinib inhibits the PI3K / AKT pathway and activates autophagy to reverse drug resistance in H1975 / OR cells; (A) KEGG pathway enrichment analysis; (B) Western blotting to detect the expression of PI3K / AKT pathway-related proteins 24 hours after drug induction; Figure 9 :Detection of the activity of baricitinib combined with osimertinib in reversing drug resistance; Figure 10 : The interaction of feizotinib with PIK3CA and DUT; (A) The intersection of disease and drug-affected proteins was combined with the target results predicted by the website to determine the possible active targets; (B) The docking experiment predicted the binding sites of feizotinib with PIK3CA, DUT and their corresponding inhibitors and target proteins; (C) Western blotting was used to detect protein expression levels; Figure 11 : Feizhuotinib accelerates the degradation of target proteins PIK3CA and DUT; (A) Cells were treated with Feizhuotinib and CHX (10 µg / ml), and the cells were harvested at the indicated times for western blot analysis of the indicated proteins; (B) Western blot analysis of target protein expression levels in cells pretreated with MG132 (10 µM) for 2 h followed by Feizhuotinib, or pretreated with CQ (20 µM) for 24 h followed by Feizhuotinib. Figure 12 : Regulatory effects of PIK3CA and DUT; (A) Western blotting was used to detect the expression levels of the indicated proteins in cells treated with TAS-114 (50 μM) for 48 h; (B) Western blotting was used to detect the expression levels of the indicated proteins in cells treated with PIK3CA-specific siRNA; Figure 13 : Overexpression of PIK3CA mediates acquired resistance to osimertinib; (A) Western blotting was used to detect the expression levels of PIK3CA and p-PIK3CA in osimertinib-sensitive and -resistant cells; (B) CCK-8 assay was used to detect the effects of osimertinib, PIK3CA-specific siRNA, or both on the proliferation of resistant cells. Figure 14 : Western blotting was used to determine the expression of the indicated proteins in cells treated with PIK3CA-specific siRNA; Figure 15: Firotinib targets DUT to inhibit cell cycle progression; (A) Western blotting was used to detect the levels of DUT in osimertinib-sensitive and -resistant cells; (B) CCK-8 assay was used to detect the effects of osimertinib, TAS-114, or both on the proliferation of resistant cells; Figure 16 : Cell cycle detection after treatment with osimertinib, si-PIK3CA, TAS-114, etc.;* p <0.05,**** p <0.0001; Figure 17 : Felotinib targets DUT to induce DNA damage; (AB) Immunofluorescence shows the effect of inhibiting DUT on DNA damage, scale bar: 200 μM; Figure 18 : In vivo antitumor efficacy of the combination of fuzotinib and osimertinib; (A) Appearance of nude mice and tumors 21 days after administration (partial display); (BC) Tumor volume and weight were measured 21 days after treatment (n=5); (D) Body weight was monitored every 3 days; Figure 19 Immunohistochemistry was used to detect the expressions of Ki-67, PIK3CA, p-PIK3CA, DUT, LC3, Bcl-2, CDK4, and p-AKT in tumor-bearing tissues of mice in different treatment groups. Flow cytometry was used to detect cell apoptosis. Scale bar: 100 μM. DETAILED DESCRIPTION

[0022] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0023] In a first aspect, the present invention provides the use of feizotinib in the preparation of a PIK3CA protein inhibitor.

[0024] The inventors have discovered that Fidotinib promotes the degradation of PIK3CA by activating the autophagy-lysosome pathway, and therefore can be used as an inhibitor of PIK3CA protein.

[0025] In a second aspect, the present invention provides the use of feizhuotinib in the preparation of DUT protein inhibitors.

[0026] The inventors have found that Fizotinib mediates DUT degradation through the ubiquitin-proteasome pathway and can therefore be used as an inhibitor of DUT proteins.

[0027] In a third aspect, the present invention provides the use of feizhuotinib in the preparation of a reversal drug for reversing acquired resistance to osimertinib in lung cancer.

[0028] The inventors have discovered that Fidotinib can target PIK3CA and then regulate the PI3K / AKT pathway to induce autophagy. By targeting DUT, it induces G1 phase arrest and mitochondrial DNA damage in cells, thereby reversing the osimertinib resistance of lung cancer.

[0029] In some optional embodiments, the lung cancer comprises non-small cell lung cancer.

[0030] In a fourth aspect, the present invention provides the use of fuzetinib combined with osimertinib in the preparation of a medicament for treating lung cancer.

[0031] The inventors have discovered that the combined use of fitrazolidinib and osimertinib can further improve the therapeutic effect of lung cancer.

[0032] In some optional embodiments, the lung cancer includes non-small cell lung cancer with acquired resistance to osimertinib.

[0033] In a fifth aspect, the present invention provides a drug for treating lung cancer, comprising fuzetinib and osimertinib.

[0034] The drug can be used to treat lung cancer, especially non-small cell lung cancer, and has a good therapeutic effect.

[0035] In some optional embodiments, the drug further comprises pharmaceutical excipients.

[0036] In some optional embodiments, the pharmaceutical excipients include but are not limited to fillers, binders, disintegrants, lubricants, flavoring agents or preservatives.

[0037] Those skilled in the art can select the types of fillers, binders, disintegrants, lubricants, flavoring agents and preservatives according to their needs.

[0038] The dosage form of the drug can be various. In some optional embodiments, the dosage form of the drug includes but is not limited to oral dosage forms.

[0039] In some optional embodiments, the oral dosage form is selected from tablets, capsules, granules and pills.

[0040] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0041] Example 1 1 Materials and Methods 1.1 Cells and Animals The human non-small cell lung cancer cell line NCI-H1975 (parental cell line) was purchased from Wuhan Punosai Biotechnology Co., Ltd. Four-week-old female BALB / c nude mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0042] 1.2 Materials Table 1 Reagent information .

[0043] 1.3 Experimental methods 1.3.1 Cell culture Cell recovery: First, inject 7 mL of pre-prepared RPMI-1640 medium into a sterile culture dish. Then, remove the cell preservation tube from the -80°C low-temperature storage device. To prevent frostbite, put it into disposable protective gloves and quickly transfer it to a 37°C constant-temperature water bath. Gently shake it to accelerate the thawing process. In the clean bench, use a pipette to draw 1 mL of thawed cell suspension and evenly inject it into the culture dish containing culture medium. Mix it thoroughly by gently shaking. Use an ethanol-proof marker pen to clearly record the cell line, activation time, cell batch and operator information on the surface of the culture dish. Finally, place the culture dish in a constant-temperature incubator and perform the first culture medium change after 6 hours.

[0044] Cell culture: Prepare complete RPMI-1640 medium containing 10% fetal bovine serum and 2% penicillin and streptomycin. Culture cells in a constant temperature incubator at 37°C and 5% CO2. Change the medium every other day to maintain normal cell growth.

[0045] Cell passaging: Cells should be passaged when they have grown to over 80%-90% confluence. First, pour the old culture medium from the original culture dish into a waste container. Then, add an appropriate amount of PBS buffer, gently shake the culture dish, and repeat the washing process twice. Use a pipette to remove the PBS solution after washing. Then, add an appropriate amount of trypsin and digest the cells for 1-2 minutes. Observe under a microscope. When the cells become round, add an equal volume of serum-containing culture medium to terminate the digestion process. Use a pipette to dislodge any adherent cells and transfer the cell suspension to a 15 mL centrifuge tube. Place the centrifuge tube in a centrifuge at 1000 rpm for 5 minutes. After centrifugation, pour the supernatant from the centrifuge tube into a waste container, retaining the cell pellet. Add an appropriate amount of culture medium to resuspend the cells and aliquot the cell suspension into several culture dishes according to the appropriate ratio. Label each dish with the cell name, the date of recovery, and the name of the culturer. Finally, place the dish in a constant temperature incubator.

[0046] Cell cryopreservation: Cells can be cryopreserved when they are in good condition and have grown to over 80%-90%. First, discard the original culture medium and add an appropriate amount of PBS, gently shaking, repeat twice. After washing the cells, remove the PBS with a pipette. Add an appropriate amount of trypsin and digest for 1-2 minutes. When the cells gradually change to a rounded shape, add an equal volume of serum-containing culture medium to terminate the digestion process. Use a pipette to dislodge the adherent cells and evenly disperse them in the culture medium. Transfer the prepared cell suspension to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. After centrifugation, carefully remove the supernatant, retaining the cell pellet. Subsequently, add 1 mL of cell freezing solution to the pellet, gently mix, and transfer to the cryovial. Seal the cryovial with parafilm and store at -80°C.

[0047] 1.3.2 Knockdown of PIK3CA Cells were seeded in a 6-well plate and incubated overnight in a constant temperature incubator until the cell density reached 60%-70%. The siRNA solution was prepared per well by mixing 3 μL of siRNA with 50 μL of OPTI-MEM serum-free medium and incubating at room temperature for 3 minutes. The Lipo2000 solution was prepared per well by mixing 6 μL of Lipo2000 transfection reagent with 50 μL of OPTI-MEM medium and incubating at room temperature for 3 minutes. The two solutions were then mixed in equal volumes and incubated at room temperature for 10 minutes to form the transfection complex. During this time, 1 mL of OPTI-MEM medium was added to each well of the 6-well plate. The prepared transfection complex was slowly added dropwise to each well, and the plate was gently rocked to evenly distribute the reagents. Six hours after transfection, the medium was changed to RPMI-1640 (without penicillin-streptomycin) supplemented with 10% fetal bovine serum. Depending on experimental requirements, cells can be harvested 48 or 72 hours after transfection for protein extraction or other assays.

[0048] 1.3.3 CCK-8 assay First, the cells were digested and then centrifuged to remove the supernatant and retain the cell pellet. An appropriate amount of culture medium was added to the pellet and mixed thoroughly with a pipette to prepare a cell suspension. A cell counting plate was used to accurately count the number of cells in the suspension. A 96-well plate was taken and 100 μL of the diluted cell suspension was added to each well. Three to six replicate wells were set up for each experimental group. The 96-well plate was placed in a constant temperature incubator for culture. After the cells were cultured for the required time, the corresponding drugs were added to each well for treatment. After the treatment was completed, 10 μL of CCK-8 reagent was added to each well to be tested, and after gently mixing, the 96-well plate was placed in a constant temperature incubator again for continued culture. After 2 h, the absorbance value of each well was detected at a wavelength of 450 nm using an enzyme reader and the obtained data was recorded.

[0049] 1.3.4 EdU assay Culture an appropriate number of cells in a 6-well plate overnight. After the cells have recovered to normal levels, perform the desired drug treatment. Prepare a 2× EdU working solution (20 μM) by diluting a 10 mM EdU solution 1:500 in cell culture medium. Add an equal volume of the 2× EdU working solution, preheated at 37°C, to the 6-well plate to achieve a final EdU concentration of 1×. Incubate the cells at 37°C for another 2 hours, then remove the culture medium and add 1 mL of paraformaldehyde solution to each well. Fix the cells at room temperature for 15 minutes to maintain cell morphology. Remove the fixative and wash the cells three times with 1 mL of wash solution per well for 3-5 minutes each to remove residual fixative. Remove the wash solution and add 1 mL of PBS containing 0.3% Triton X-100 per well. Incubate at room temperature for 10-15 minutes to increase cell membrane permeability. Remove the permeabilization solution and wash the cells one to two times with 1 mL of wash solution per well for 3-5 minutes each to ensure removal of residual reagent. Remove the wash buffer and add 0.5 mL of Click reaction solution to each well. Gently shake the plate to evenly cover the sample. Incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash three times with wash buffer for 3-5 minutes each to remove unreacted reagent. Add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes to stain the nuclei. Aspirate the Hoechst 33342 solution and wash three times with wash buffer for 3-5 minutes each to remove residual dye. After completing these steps, perform fluorescence microscopy to observe and record the experimental results.

[0050] 1.3.5 Clone formation assay Digest the cells, then centrifuge, remove the supernatant, and retain the cell pellet. Add an appropriate amount of culture medium to the pellet and mix thoroughly to prepare a cell suspension. Accurately count the cells in the suspension using a cell counting plate and add them to a 6-well plate to ensure an appropriate cell density. Discard the original culture medium and wash 1-2 times with an appropriate amount of PBS. Discard the PBS and fix with paraformaldehyde for 20 minutes. Discard the paraformaldehyde and wash 1-2 times with an appropriate amount of PBS. Discard the PBS and stain with an appropriate amount of crystal violet for 30 minutes. Discard the crystal violet and wash 1-2 times with PBS. Allow to dry and then photograph.

[0051] 1.3.6 Scratch test Digest the cells and centrifuge them. Discard the supernatant, retain the pellet, add an appropriate amount of culture medium, pipette evenly, and count the cells to obtain a cell suspension of appropriate density. Plate the cells in a 6-well plate and incubate in a constant temperature incubator. Once the cells have grown confluently, use a ruler to guide the cells while moving a sterile 20 μL pipette tip from one end of the well to the other, keeping the tip vertical and not tilted. Discard the old culture medium and gently rinse with PBS until all the cells are removed. Add drug treatment according to the experimental protocol and incubate the cells in a constant temperature incubator. Remove the 6-well plate after 0, 12, and 24 hours, observe under a microscope, and photograph.

[0052] 1.3.7 Cell cycle assay Culture an appropriate number of cells in a 6-well plate. After the cells adhere and return to normal, treat the cells according to the experimental protocol; discard the original culture medium, wash the adherent cells once with PBS, and add an appropriate amount of trypsin to digest the cells; add RPMI-1640 culture medium containing serum, gently pipette the cells off, transfer them to a centrifuge tube, centrifuge at 1000 g for 5 minutes, discard the supernatant, retain the cell pellet, add an appropriate amount of PBS to gently resuspend the cells and count; collect approximately 5×10 5 Cells were centrifuged for 5 min and the supernatant was discarded; 1 mL of PBS was added to wash, centrifuged for 5 min and the supernatant was discarded; 0.3 mL of PBS was added to resuspend the cells, and then 1.2 mL of anhydrous ethanol stored at -20°C overnight was added, mixed thoroughly and placed in a -20°C refrigerator overnight; centrifuged for 5 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the cells, and the cells were placed at room temperature for 15 min; centrifuged for 5 min, the supernatant was discarded, 100 μL of RNase A Reagent was added to fully suspend the cells, and the cells were incubated in a 37°C water bath for 30 min; 400 μL of PI Reagen was added and mixed thoroughly, and the cells were incubated at 2-8°C in the dark for 30 min and immediately tested.

[0053] 1.3.8 Cell apoptosis assay An appropriate number of cells were cultured in a 6-well plate. After the cells adhered and returned to normal, the required drug treatment was performed. The cell culture medium was aspirated into a centrifuge tube, the adhered cells were washed once with PBS, and an appropriate amount of trypsin was added to digest the cells. The collected cell culture medium was added, the cells were gently blown off, transferred to a centrifuge tube, centrifuged for 5 min, the supernatant was discarded, the cell pellet was retained, and the cells were gently resuspended in an appropriate amount of PBS and counted. 100,000 resuspended cells were taken, centrifuged for 5 min, the supernatant was discarded, and 195 μL Annexin V-FITC binding solution was added to gently resuspend the cells. 5 μL Annexin V-FITC was added and gently mixed. 10 μL propidium iodide staining solution was added and gently mixed. The cells were incubated at room temperature in the dark for 20 min, then placed in an ice bath and tested on a microscope.

[0054] 1.3.9 Western Blot (WB) Protein extraction and preparation of protein samples: discard the original culture medium, add pre-cooled PBS, wash three times, discard and aspirate clean; add lysis buffer, scrape the cells with a cell scraper on ice, collect them into a clean EP tube, mark them, and lyse them on ice for 30 min; pre-cool the centrifuge to 4°C in advance, put in the EP tube, balance it, and centrifuge at 12000 rpm at 4°C for 30 min; collect the supernatant into another clean and labeled EP tube; take part of the supernatant for dilution, blow it evenly with a pipette, take 20 μL of the diluted protein standard solution of different concentrations and the protein sample to be tested and add them to a 96-well plate in turn; add 200 μL of BCA working solution to each well, gently shake the 96-well plate to mix the solution, and incubate at 37°C for 30 min; use a microplate reader to measure the absorbance at 562 nm. A standard curve was drawn with the concentration of the protein standard as the horizontal axis and the absorbance as the vertical axis. The concentration of the corresponding protein sample was calculated based on the measured absorbance value of the protein sample to be tested. The protein loading volume V (V = 30 / C) was calculated based on a total amount of 30 μg of protein. An appropriate volume of 5× protein loading buffer was added to a final concentration of 1×. The liquid was vortexed to mix thoroughly, and the sample was boiled at 100°C for 10 min. After the sample cooled to room temperature, it was stored at -20°C.

[0055] Electrophoresis: Vortex the protein sample to mix thoroughly; add an appropriate amount of electrophoresis buffer to the electrophoresis tank; slowly add protein molecular weight standards and protein samples to the sample wells according to the calculated results; maintain a constant voltage of 80 V for 30 minutes, then switch to a constant voltage of 120 V for approximately 40 minutes.

[0056] Transfer: Soak the cut PVDF membrane in anhydrous methanol to fully activate it and set aside; lay the white side of the clamp flat, place a sponge and filter paper on it, soak them together in the transfer solution, and then place the membrane on the filter paper; carefully pry open the plastic plate, cut off the excess gel, carefully place the gel on the membrane, taking care to avoid bubbles, and cover the gel with the filter paper and sponge soaked in transfer solution in turn; place the clamped clamp in the transfer tank, place ice cubes around the transfer tank to cool it down, and set the transfer conditions according to the molecular weight of the target protein, generally constant voltage 80 V, 90 min.

[0057] Blocking: Place the transferred PVDF membrane in 5% skim milk powder and block on a shaker at room temperature for 2 h.

[0058] Incubation with primary antibody: After blocking, cut the membrane according to the experimental protocol and place the cut membrane in primary antibody diluent (1:1000) prepared with 5% skim milk powder in a 4°C refrigerator overnight.

[0059] Incubation with secondary antibody: Recover the primary antibody and place it in a 4°C refrigerator. Add TBST and wash three times on a shaker. Add the appropriate amount of secondary antibody dilution (1:5000) prepared in TBST and incubate on a shaker at room temperature for 1 h.

[0060] Luminescence detection: Recover the secondary antibody and place in a 4°C refrigerator. Add TBST and wash three times on a shaker. Add the prepared ECL luminescent solution to the membrane, place in a gel imaging system, and expose for imaging.

[0061] 1.3.10 Immunofluorescence Cells at an appropriate density were seeded in a 6-well plate and cultured overnight for drug treatment; the original culture medium was discarded and an appropriate amount of PBS was added along the wall of the dish to wash 1-2 times to prevent washing away the cells; after adding paraformaldehyde for fixation for 25 minutes, the paraformaldehyde was discarded and the plate was washed 3 times with an appropriate amount of PBS; an appropriate amount of prepared 0.2% TritonX-100 was added and the plate was permeabilized on a shaker for 1 hour and then discarded and washed 3 times with an appropriate amount of PBS; an appropriate amount of prepared goat serum was added and blocked for 1 hour; the goat serum was discarded and the primary antibody solution diluted with fluorescent antibody diluent was added and the plate was refrigerated at 4°C overnight; the primary antibody was recovered and the plate was washed 3 times with an appropriate amount of PBS; the prepared secondary antibody solution was added and incubated in a 37°C incubator in the dark for 1 hour. From this step onwards, all remaining operations need to be protected from light; the secondary antibody solution was discarded and the plate was washed 3 times with an appropriate amount of PBS; the prepared DAPI solution was added and incubated at room temperature for 3 minutes; the plate was washed 3 times with PBS and observed under an inverted fluorescence microscope.

[0062] 1.3.11 Confocal laser scanning microscopy analysis H1975 / OR cells were transfected with adenovirus carrying the mRFP-GFP-LC3 fusion protein. After treatment with the indicated drugs for 48 h, the cells were observed using a Leica TCS SP8 laser scanning confocal microscope.

[0063] 1.3.12 Molecular docking Virtual docking of target proteins and small molecule compounds was performed using Autodock 4.2. The 3D structures of PIK3CA (PDB ID: 5H4J) and DUT (PDB ID: 6OAC) were optimized. The compounds were virtually docked onto the target proteins. Based on the docking scores, the highest-scoring group was selected and imported into PyMoL for visualization.

[0064] 1.3.13 Kyoto Encyclopedia of Genes and Genomes (KEGG) Analysis Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using Microinformatics (https: / / www.bioinformatics.com.cn / ).

[0065] 1.3.14 Animal experiments Prepare cells: Culture cells in a culture dish to a density of 90%; add an appropriate amount of PBS to the culture dish, wash twice, add an appropriate amount of trypsin, and digest for 2 min. Add an equal volume of serum-containing medium to the trypsin to terminate digestion. Pipette the cell suspension into a 15 mL centrifuge tube and centrifuge. After centrifugation, discard the supernatant, resuspend the cells in pre-chilled PBS, and centrifuge. After centrifugation, discard the supernatant, add Matrigel and pre-chilled PBS in a 1:1 ratio, mix well, transfer to an EP tube, and place on crushed ice until used.

[0066] Subcutaneous tumor implantation: Each mouse was ear-tagged and recorded; the injection site was disinfected with iodine, and the cell suspension was blown evenly before injection. Each mouse was injected with 100 μL of cell suspension.

[0067] Observation and drug administration: After tumor formation, mice were randomly divided into groups and drug administration was performed every other day. The volume of subcutaneous tumors was measured with a vernier caliper. The calculation formula was: V = (length * width 2 ) / 2, measure the weight of the mice and keep a record, and observe whether the activities of the nude mice are normal.

[0068] Tumor removal: When the drug administration period expires and the volume of the subcutaneous tumor of the mouse is within the ethical range, the mouse is killed, the subcutaneous tumor is isolated, and fixed in paraformaldehyde.

[0069] 1.3.15 HE staining Immerse in xylene three times, 10 min each time; immerse in anhydrous ethanol three times, 5 min each time; immerse in 95%, 85%, and 75% ethanol for 5 min each, and wash three times with PBS; stain with hematoxylin for 3 min, wash with PBS for 1 min; differentiate with differentiation solution for 2 min, rinse with running water for 2 min; stain with eosin for 1 min, wash with PBS for 1 min; immerse in 75%, 85%, and 95% ethanol for 3 min each; immerse in anhydrous ethanol three times, 3 min each time; immerse in xylene three times, 3 min each time; add neutral gum to seal the slides, let them dry, and observe under an upright biological microscope.

[0070] 1.3.16 Immunohistochemical staining Immerse in xylene 3 times, 10 min each time; immerse in anhydrous ethanol 3 times, 5 min each time; immerse in 95%, 85%, and 75% ethanol for 5 min each, wash 3 times with PBS; boil sodium citrate repair solution in a microwave oven, put in paraffin sections, heat for 1 min on medium heat and 3 min on low heat, perform 4 cycles, and then let it return to room temperature naturally; draw circles with a tissue pen, add endogenous peroxidase blocker, and incubate at room temperature for 10 min; wash 3 times with PBS, wipe dry with filter paper; add primary antibody and refrigerator at 4°C overnight; wash 3 times with PBS, add reaction enhancement solution and incubate at 37°C for 20 min; wash 3 times with PBS, add enhanced enzyme-labeled goat anti-rabbit IgG polymer and incubate at 37°C for 20 min; wash 3 times with PBS, add appropriate amount of freshly prepared DAB color development solution and observe the degree of color development under a microscope; rinse with running water for 10 min, add hematoxylin and stain for 45 s; rinse with running water for 1 min, add slow differentiation solution, differentiate for 25 s, rinse with running water for 1 min; immerse in 75%, 85%, and 95% ethanol for 3 min in sequence; immerse in anhydrous ethanol three times, 3 min each time; immerse in xylene three times, 3 min each time; add neutral gum to seal the slides, let them dry, and observe under an upright biological microscope.

[0071] 1.3.17 Statistical Analysis Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). Data are expressed as mean ± SD. Multiple comparisons were performed using one-way ANOVA. p <0.05 indicates that the difference is statistically significant ("*" indicates p <0.05, “**” indicates p <0.01, “***” indicates p <0.001, “****” indicates p <0.0001).

[0072] 2 Results 2.1 Construction of drug-resistant cell model In order to quickly construct an acquired drug-resistant cell model of lung cancer, H1975 cells were induced to continuously be exposed to osimertinib using a concentration gradient method, and the acquired drug-resistant strain H1975 / OR ( Figure 1 A in Figure 1). Optical microscopy showed that H1975 / OR cells were larger in size and had a higher proportion of spindle-shaped morphology than parental cells ( Figure 1 The dose-effect curves calculated based on CCK-8 assay showed that the half-maximal inhibitory concentration (IC) of osimertinib for H1975 cells and H1975 / OR cells was 100%.50 ) were 3.2 μM and 12.1 μM ( Figure 1 C in Figure ). To further explore the differences between the H1975 cell line exposed to osimertinib and its osimertinib-resistant cell line (H1975 / OR), a cell apoptosis assay was performed. The results showed that in the medium with a higher concentration of osimertinib, the apoptosis rate of the resistant cells was significantly lower than that of the parental cells ( Figure 1 The above data indicate that the drug-resistant cell line established in this study can be used as a cell line model for studying drug resistance mechanisms and screening anti-drug resistance activity.

[0073] 2.2 Felotinib enhances the sensitivity of drug-resistant cells to osimertinib This study, based on a drug screening platform for anti-resistance activity, found that the JAK2 inhibitor feizotinib has the activity to reverse EGFR-TKI resistance in lung cancer. To explore the combined effect of feizotinib and osimertinib on H1975 / OR cells, CCK-8 assay showed that compared with the osimertinib (Fdtn) monotherapy group, the combined use of feizotinib significantly weakened the proliferation ability of H1975 / OR cells ( Figure 2 Similarly, the results of EdU experiment and colony formation experiment also showed similar phenomena ( Figure 2 In addition, the scratch test also found that the combined treatment of fuzotinib and osimertinib (Osim) can effectively reduce the migration ability of H1975 / OR cells ( Figure 2 These results suggest that feizotinib can restore the sensitivity of drug-resistant cells to EGFR-TKIs, and that feizotinib combined with osimertinib has an anti-resistant tumor effect.

[0074] 2.3 Felotinib induces G1 cycle arrest To further explore the inhibitory effect of feizotinib on H1975 / OR cell proliferation, flow cytometry was used to detect the cell cycle distribution of H1975 / OR cells treated with feizotinib. The results showed that feizotinib treatment could significantly induce cell cycle arrest in H1975 / OR cells at the G1 phase ( Figure 3 A in Figure 1). Using WB, we further verified the effect of Fidotinib on cell cycle-related factors. Figure 3 As shown in Figure B, the combination of feizotinib and osimertinib significantly increased the proportion of H1975 / OR cells in the G1 phase and reduced the expression level of the cyclin CDK4. These results indicate that feizotinib can regulate the cell cycle of H1975 / OR cells and induce G1 cell arrest.

[0075] 2.4 Felotinib regulates the PI3K / AKT pathway to induce autophagy and reverse drug resistance 2.4.1 Felotinib combined with osimertinib induces apoptosis in drug-resistant cells Given that apoptosis plays a key role in the anti-proliferative mechanism mediated by anticancer drugs, this study first examined the effect of feizotinib on apoptosis in H1975 / OR cells. Flow cytometry results showed that the combination of feizotinib and osimertinib significantly increased the apoptosis level of H1975 / OR cells compared with feizotinib alone ( Figure 4 However, WB analysis did not detect significant changes in apoptosis marker proteins such as Bcl-2 and BAX ( Figure 4 The above experimental data suggest that the enhanced sensitivity of resistant cells to osimertinib by fizotinib may not be dependent on inducing apoptosis, and it is possible that other mechanisms may play a role in its anti-resistance activity.

[0076] 2.4.2 Felotinib induces autophagy in drug-resistant cells Autophagy is an important channel for cancer cells to acquire drug resistance and promote tumor progression. This study used Western blotting and immunofluorescence to detect autophagy-related markers to evaluate the drug-induced autophagy. The results showed that after administration of Fizotinib, the expression level of the autophagy-related protein LC3-Ⅱ in H1975 / OR cells was significantly upregulated ( Figure 5 A and B in the figure).

[0077] The effect of filgotinib on autophagic flux was assessed using mRFP-GFP-LC3. Figure 6 The number of yellow granules (representing autophagosomes before fusion with acidic lysosomes) in the feizotinib group increased significantly. These results indicate that feizotinib can effectively activate the autophagy process in H1975 / OR cells, suggesting that autophagy may play an important role in feizotinib-mediated anti-resistance activity.

[0078] 2.4.3 Felotinib activates autophagy to reverse drug resistance To further investigate the role of autophagy in the inhibition of H1975 / OR cell proliferation by fuzotinib, this study used the autophagy inhibitor chloroquine (CQ) for intervention experiments. Flow cytometry was used to detect cell apoptosis levels, and CCK-8 assays were used to measure cell viability. The results showed that the apoptosis rate of H1975 / OR cells in the CQ-treated group was significantly reduced, while the cell proliferation ability was significantly restored ( Figure 7 The above experimental results suggest that feizotinib activates autophagy in H1975 / OR cells, thereby reversing the drug resistance of tumor cells.

[0079] 2.4.4 Felotinib inhibits the PI3K / AKT pathway to activate autophagy and reverse drug resistance To explore the mechanism of drug-activated autophagy, the signal transduction process of Fizotinib was further analyzed based on KEGG pathway enrichment, which showed that it was significantly enriched in the PI3K / AKT signaling pathway ( Figure 8 To verify the prediction, the protein level was tested. Figure 8 As shown in Figure B, Western blot analysis confirmed that treatment with Fizotinib significantly downregulated the phosphorylation levels of key proteins in the PI3K / AKT pathway. These results suggest that Fizotinib negatively regulates the PI3K / AKT signaling pathway, thereby activating autophagy.

[0080] 2.5PIK3CA and DUT are potential targets for fuzotinib anti-tumor resistance 2.5.1 The anti-resistance activity of fuzotinib is unrelated to its JAK2 targeting effect Because feizotinib is a traditional JAK2 inhibitor, and JAK2 is a key target for myeloproliferative diseases, it is also considered an important target in the field of solid tumors such as breast cancer. In addition, studies have shown that JAK2 is closely related to the resistance of esophageal cancer cells to cisplatin. Based on these findings, in the process of studying its anti-resistance activity targets, the hypothesis was first proposed: feizotinib may reverse osimertinib resistance by targeting JAK2 in H1975 / OR cells. To verify this possibility, the effect of another JAK2 inhibitor, baricitinib, on osimertinib-resistant cells was evaluated. Figure 9 As shown in the results, baricitinib does not have the effect of reversing drug resistance. This result suggests that the anti-resistance activity of feizotinib may not be dependent on its classic target JAK2, and further exploration of other possible molecular targets of feizotinib and its mechanism of action is needed.

[0081] 2.5.2 PIK3CA and DUT were found to be potential targets of fuzetinib In order to find the anti-tumor resistance target of feizotinib, we first screened the potential targets of feizotinib and its related diseases using bioinformatics methods, and used a cross-analysis strategy to obtain common targets. Database analysis showed that PIK3CA and DUT may be potential targets of feizotinib ( Figure 10 A in Figure 1). PIK3CA is one of the most commonly mutated genes in solid cancers, and its mutation has been shown to mediate EGFR-TKI resistance. Furthermore, overexpression of DUTs in various tumors has also been widely reported. To further validate the specific binding of pizotinib to the above target proteins, this study employed molecular docking analysis. Figure 10As shown in Figure B, molecular docking results show that feizotinib binds significantly better to PIK3CA than its known inhibitor, apellix, and the binding sites of the two are different. Similarly, feizotinib binds to DUT better than its known inhibitor, TAS-114, and the binding regions and binding sites of the two are also different. In addition, feizotinib can significantly inhibit the protein expression of PIK3CA and DUT, and the inhibitory effect is more pronounced when used in combination with osimertinib ( Figure 10 C in the figure). Based on the above experimental results, PIK3CA and DUT may be the targets of the anti-resistance activity of feizotinib, which provides a direction for further elucidating the anti-tumor targets of feizotinib.

[0082] 2.5.3 Felotinib regulates protein expression of PIK3CA and DUT After discovering that Fizotinib can inhibit the protein expression of target proteins PIK3CA and DUT, we further explored its mechanism of regulating target proteins. We used the protein synthesis inhibitor cycloheximide (CHX) to treat the cells and evaluated the effect of Fizotinib on the protein degradation rate of PIK3CA and DUT through protein stability experiments. The experimental results showed that after CHX administration, the Fizotinib group significantly accelerated the protein degradation rate of PIK3CA and DUT in H1975 / OR cells ( Figure 11 (A in Figure 1). This suggests that feizotinib regulates the expression of PIK3CA and DUT by accelerating protein degradation. To further elucidate the mechanism of feizotinib-mediated protein degradation, this study evaluated the role of the autophagy-lysosome system and the ubiquitin-proteasome pathway. Intervention experiments using the proteasome-specific inhibitor MG132 and the autophagy inhibitor CQ showed that MG132 treatment reversed the inhibitory effect of feizotinib on PIK3CA expression, while having no effect on DUT expression. CQ treatment maintained the inhibitory effect of feizotinib on PIK3CA expression, while reversing the inhibitory effect on DUT expression. Figure 11 These results indicate that Fidotinib regulates the expression of PIK3CA through autophagy and lysosomes and DUT through ubiquitination.

[0083] To investigate whether there is a regulatory relationship between the target protein PIK3CA and DUT, H1975 / OR cells were treated with PIK3CA-specific siRNA and the DUT-specific inhibitor TAS-114, respectively. Western blot results showed that under conditions of PIK3CA gene silencing, the expression levels of PIK3CA and its phosphorylated form (p-PIK3CA) were significantly reduced, while the protein expression level of DUT did not change significantly ( Figure 12A in Figure 3) indicates that DUT expression is independent of PIK3CA regulation. Similarly, under TAS-114-mediated DUT inhibition, DUT protein expression was significantly downregulated, but the expression levels of PIK3CA and its phosphorylated form remained stable ( Figure 12 (B) indicates that PIK3CA expression is independent of DUT regulation. These experimental data demonstrate that PIK3CA and DUT do not regulate each other at the protein level, suggesting that these two targets may play independent roles in the resistance-reversal activity of feizotinib, making feizotinib a dual-target inhibitor of PIK3CA and DUT.

[0084] 2.6 Felotinib targets PIK3CA to activate autophagy and reverse drug resistance 2.6.1 PIK3CA Overexpression Mediates Acquired Resistance to Osimertinib To clarify the role of PIK3CA in H1975 / OR cells, this study first quantitatively analyzed its expression level by Western blotting. The experimental results showed that the total protein level and the expression of its phosphorylated form of PIK3CA were upregulated in H1975 / OR drug-resistant cells compared with the parental control cells ( Figure 13 A in ). Figure 13 As shown in Figure B, CCK-8 assays showed that downregulation of PIK3CA expression inhibited the proliferation activity of H1975 / OR cells. Inhibition of PIK3CA and administration of osimertinib reversed the resistance. These data demonstrate that overexpression of PIK3CA in H1975 / OR cells mediates acquired resistance to osimertinib.

[0085] 2.6.2 Felotinib targets PIK3CA to activate autophagy and reverse drug resistance To elucidate the role of PIK3CA in acquired resistance to osimertinib, cells were treated with PIK3CA siRNA. Western blot results showed that PIK3CA gene silencing significantly reduced the expression levels of AKT and its phosphorylated form (p-AKT), accompanied by upregulation of the autophagy marker LC3-II ( Figure 14 These results suggest that inhibition of PIK3CA can activate the cellular autophagy process, based on the detected inhibition of PI3K / AKT pathway activity by pizotinib ( Figure 8 B), suggesting that feizotinib regulates PIK3CA to induce autophagy through the PI3K / AKT signaling pathway, thereby reversing the resistance of tumor cells to osimertinib.

[0086] 2.7 Felotinib targets DUT to arrest cell cycle and induce mitochondrial damage 2.7.1 Overexpression of DUT Mediates Acquired Resistance to Osimertinib To clarify the biological function of DUT in H1975 / OR cells, the expression level of DUT was quantitatively analyzed by Western blotting. The experimental results showed that the protein expression level of DUT in H1975 / OR resistant cells was significantly upregulated compared with that in parental H1975 cells ( Figure 15 To further explore the regulatory role of DUT in cell proliferation, H1975 / OR cells were treated with the DUT-specific inhibitor TAS-114. The results of the cell proliferation experiment showed that inhibiting the expression of DUT could significantly reduce the proliferation ability of H1975 / OR cells ( Figure 15 These results suggest that overexpression of DUT in H1975 / OR cells is one of the important mechanisms mediating acquired resistance to osimertinib, and DUT may become a potential therapeutic target for overcoming osimertinib resistance.

[0087] 2.7.2 Felotinib targets DUT to induce cell cycle G1 arrest Previous studies have found that DUTs play an important role in cell cycle regulation and can induce cell cycle arrest. To investigate whether the G1 arrest induced by pizotinib in H1975 / OR cells is related to its targeting of DUTs, flow cytometry was used for cell cycle analysis. The experimental results showed that the combination of TAS-114 and osimertinib synergistically induced a more significant G1 arrest compared to the combination of PIK3CA gene silencing and osimertinib treatment ( Figure 16 ), suggesting that DUT plays a key role in cell cycle regulation and that the cell cycle arrest activity of feizotinib is related to its targeting of DUT.

[0088] 2.7.3 Felotinib targets DUT-induced mitochondrial DNA damage in drug-resistant cells Previous literature has reported that DUT is closely associated with DNA damage response. This study first detected the nuclear DNA damage marker γH2AX through immunofluorescence staining and found that there was no difference in the expression of γH2AX among the groups ( Figure 17 A in the figure). The expression level of mitochondrial DNA damage marker TFAM was then evaluated. Experimental data showed that the expression of TFAM was significantly upregulated in the group treated with fuzetinib and osimertinib ( Figure 17 (B) A similar trend was observed in the TAS-114 combined with osimertinib group, but not in the si-PIK3CA combined with osimertinib group, indicating that Fizotib induces mitochondrial DNA damage by targeting DUT, thereby reversing osimertinib resistance.

[0089] In summary, feizotinib exerts a dual mechanism of action by targeting DUTs: on the one hand, it inhibits tumor cell proliferation by regulating G1 cell cycle arrest; on the other hand, it reverses osimertinib resistance by inducing mitochondrial DNA damage. These findings provide new experimental evidence for elucidating the anti-tumor mechanism of feizotinib and lay a theoretical foundation for the further development of anti-tumor therapeutic strategies based on DUT targets.

[0090] 2.8 In vivo experiments To evaluate the in vivo effects of feizotinib combined with osimertinib on H1975 / OR cells, H1975 / OR cells were injected subcutaneously into nude mice to establish a mouse xenograft model. Mice were randomly assigned to four groups and given feizotinib (5 mg / kg), osimertinib (10 mg / kg), a combination of the two drugs, or saline as a control. Results from the H1975 / OR nude mouse xenograft model showed that the combination of feizotinib and osimertinib significantly inhibited tumor growth compared to osimertinib alone, with no significant toxicity observed ( Figure 18 AD in ).

[0091] Immunohistochemistry assay showed that the expression of PIK3CA, p-PIK3CA, DUT, Bcl-2, CDK4, and p-AKT was significantly decreased, and the expression of LC3 was significantly increased in the fuzetinib group and the combination group ( Figure 19 These results were consistent with the results of in vitro mechanistic studies, except for Bcl-2. These results indicate that combination therapy exhibited a stronger antitumor effect than single-agent therapy in H1975 / OR xenograft mice.

[0092] 3 Summary EGFR-TKIs, as the first-line standard treatment for patients with advanced NSCLC harboring activating EGFR mutations, have significantly improved survival outcomes. However, their clinical application is still limited by the development of acquired resistance. Although the third-generation EGFR-TKI osimertinib has effectively overcome some resistance issues by targeting the T790M mutation, resistance remains a major obstacle to its long-term efficacy. Therefore, developing therapeutic strategies that can delay or reverse osimertinib resistance is of great clinical value.

[0093] This study found that the JAK2 inhibitor feizotinib can effectively reverse osimertinib resistance and enhance the inhibitory effect of osimertinib on resistant cells. The combination of feizotinib and osimertinib demonstrated robust antitumor activity in both in vitro and in vivo studies. Although feizotinib has been widely reported to be effective in myeloproliferative neoplasms, its role in solid tumors, particularly EGFR-TKI-resistant NSCLC, has been underexplored. To further investigate the mechanisms underlying osimertinib resistance, we successfully established an osimertinib-resistant H1975 / OR cell line using a dose-escalation approach. This study demonstrated that the combination of feizotinib and osimertinib significantly inhibited the proliferation, invasion, and migration of resistant cells. This result echoes previous reports demonstrating that feizotinib combined with erlotinib can reverse drug resistance. This finding also provides important evidence for the novel application of feizotinib. Further mechanistic studies revealed that feizotinib exerts its resistance-reversing effect through a dual mechanism of action: inhibiting tumor cell proliferation by inducing G1 cell cycle arrest and promoting the clearance of resistant cells by activating the autophagy-dependent cell death pathway. Notably, the autophagy inhibitor chloroquine (CQ) partially attenuated the antiproliferative effect of the combination therapy, suggesting that autophagy is the primary pro-death mechanism in this model. Although feizotinib has been well-established as a highly selective JAK2 inhibitor, the introduction of another highly potent JAK2 inhibitor, baricitinib, failed to replicate the resistance-reversing effect, suggesting that feizotinib's effects may be independent of the canonical JAK2 signaling pathway and suggest the existence of a novel molecular target.

[0094] By integrating bioinformatics analysis with experimental validation, this study identified PIK3CA and DUT as key targets for osimertinib in reversing drug resistance. Molecular docking and protein stability experiments demonstrated that osimertinib regulates these two targets through distinct protein degradation pathways: PIK3CA degradation is promoted by activating the autophagy-lysosome pathway, while DUT degradation is mediated by the ubiquitin-proteasome pathway. Notably, PIK3CA and DUT are functionally independent, with neither upstream-downstream regulatory relationships nor synergistic expression patterns, suggesting that osimertinib reverses drug resistance through a dual-target mechanism. This finding provides new insights into multi-target drug development. Further studies revealed that overexpression of PIK3CA in osimertinib-resistant cells is a key mechanism of resistance to osimertinib. By targeting PIK3CA, osimertinib significantly inhibited the PI3K / AKT signaling axis, manifested by a simultaneous downregulation of PIK3CA, p-PIK3CA, AKT, and p-AKT protein levels, accompanied by significant accumulation of the autophagy marker LC3-II. PIK3CA gene silencing experiments further confirmed this regulatory mechanism, suggesting that feizotinib may restore the sensitivity of drug-resistant cells to osimertinib by inhibiting the PI3K / AKT pathway. On the other hand, previous studies have shown that DUT is not only associated with cell cycle progression, but also closely linked to DNA damage. This study found that feizotinib induced G1 phase arrest and mitochondrial DNA damage by targeting DUT, and the DUT inhibitor TAS-114 could mimic this effect. This result not only reveals the key role of DUT in maintaining the genomic stability of drug-resistant cells, but also provides a new approach to targeting the DNA damage response pathway to overcome drug resistance. In vivo experiments further confirmed that the combination of feizotinib and osimertinib can significantly inhibit the growth of transplanted tumors without increasing systemic toxicity, highlighting its potential for clinical translation.

[0095] In summary, this study discovered a novel mechanism by which fuzotinib reverses osimertinib resistance by dually targeting PIK3CA and DUT, providing a novel therapeutic strategy for overcoming EGFR-TKI resistance. Future studies are needed to validate its efficacy in more complex preclinical models (such as drug-resistant organoids) and explore combination therapy regimens based on dual PIK3CA / DUT inhibitors to advance this discovery into clinical practice and provide candidate drugs for clinical treatment of targeted drug resistance in lung cancer, thereby improving efficacy.

[0096] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of fuzotinib in the preparation of PIK3CA protein inhibitors.

2. Application of fuzotinib in the preparation of DUT protein inhibitors.

3. Use of fuzotinib in the preparation of a reversal drug for reversing acquired resistance to osimertinib in lung cancer.

4. Use of fuzotinib combined with osimertinib in the preparation of drugs for the treatment of lung cancer.

5. The use according to claim 4, characterized in that The lung cancer includes lung cancer with acquired resistance to osimertinib.

6. A drug for treating lung cancer, characterized in that: The drugs include fuzetinib and osimertinib.

7. The drug according to claim 6, characterized in that The medicine also includes pharmaceutical excipients.

8. The drug according to claim 7, characterized in that The pharmaceutical excipients include at least one of a filler, a binder, a disintegrant, a lubricant, a flavoring agent or a preservative.

9. The drug according to claim 6, characterized in that The dosage form of the drug includes oral dosage form.

10. The drug according to claim 9, characterized in that The oral dosage form is selected from the group consisting of tablets, capsules, granules and pills.

Images