3-AT in the treatment of chronic myeloid leukemia TKIs resistance caused by BCR::ABL amplification / overexpression

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of 3-AT in the treatment of tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) caused by BCR::ABL amplification / overexpression. This invention first demonstrates that CML cells with BCR::ABL amplification / overexpression are resistant to multiple TKIs, and that catalase (CAT) protein expression and enzyme activity are elevated in these cells, suggesting that CAT is a potential therapeutic target for these drug-resistant cells. 3-Amino-1,2,4-triazole (3-AT) is an inhibitor of CAT. This invention is the first to apply 3-AT to the treatment of TKI-resistant CML caused by BCR::ABL amplification / overexpression, and confirms its effectiveness. Specifically: 3-AT can inhibit the proliferation of BCR::ABL-amplified / overexpressing CML-resistant cells; and can reduce the IC50 values ​​of four TKIs—imatinib, nilotinib, dasatinib, and ponatinib—on these CML-resistant cells, indicating that 3-AT can enhance the sensitivity of these resistant cells to multiple TKIs; 3-AT can inhibit the proliferation of bone marrow cells in CML-resistant patients and synergistically enhance the inhibitory effect of imatinib on bone marrow cells in CML-resistant patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical research technology, specifically involving the application of 3-AT in the treatment of TKI resistance in chronic myeloid leukemia caused by BCR::ABL amplification / overexpression. Background Technology

[0002] Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm (MPN) caused by the BCR::ABL fusion gene, and its clinical treatment relies on tyrosine kinase inhibitors (TKIs). However, resistance or intolerance to TKIs in CML patients is a primary challenge in clinical treatment. CML drug resistance is mainly divided into BCR::ABL-dependent resistance and BCR::ABL-independent resistance. The former can be further divided into resistance caused by BCR::ABL point mutations and resistance caused by BCR::ABL amplification / overexpression. Second- and third-generation TKIs have overcome most resistance caused by point mutations, but there is still no effective solution for resistance caused by BCR::ABL amplification / overexpression. Therefore, it is urgent to explore new therapeutic targets for this type of resistance.

[0003] Catalase (CAT) is a key enzyme in cells that scavenge reactive oxygen species (ROS). It efficiently catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen, playing a crucial role in maintaining intracellular redox balance. Recent studies have shown that CAT is highly expressed in various tumors and mediates drug resistance. For example, CAT expression is upregulated in solid tumors such as liver cancer, lung cancer, and gastric cancer. It protects tumor cells from oxidative damage by scavenging chemotherapy-induced ROS, thereby mediating chemotherapy resistance (see Glorieux C, Buc Calderon P. Targeting catalase in cancer. Redox Biol. 2024 Nov;77:103404). Increased CAT activity is also observed in hematologic malignancies and is closely related to drug resistance. 3-Amino-1,2,4-triazole (3-AT), as a CAT inhibitor, has been reported to sensitize chemotherapeutic drugs in some solid tumors by inhibiting CAT activity and blocking H2O2 clearance (see Bechtel W, Bauer G. Modulation of intercellular ROS signaling of human tumor cells. Anticancer Res. 2009 Nov;29(11):4559-70). CAT expression is elevated in BCR::ABL amplified / overexpressed CML, but whether its inhibitor 3-AT can reverse drug resistance in this type of CML by targeting and inhibiting CAT is currently unknown. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a novel strategy for reversing TKI resistance in chronic myeloid leukemia caused by BCR::ABL amplification / overexpression.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] The embodiments of the present invention provide an experimental verification of the application of 3-AT in the treatment of TKI resistance in chronic myeloid leukemia caused by BCR::ABL amplification / overexpression.

[0007] Furthermore, the 3-AT is a CAT inhibitor.

[0008] Furthermore, the drug is applied to chronic myeloid leukemia with BCR::ABL amplification / overexpression, wherein the drug resistance is resistance to imatinib, dasatinib, nilotinin, and ponatinib.

[0009] Furthermore, the drug significantly reduces the IC50 value of TKIs by inhibiting CAT enzyme activity.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] (1) This invention is the first to discover that 3-AT can effectively reverse BCR::ABL amplification / overexpression-mediated CML resistance, providing a new treatment strategy and experimental basis for overcoming this type of resistance.

[0012] (2) This invention verified that 3-AT can reduce the IC50 values ​​of four TKIs, namely imatinib, nilotinib, dasatinib and ponatinib, to this type of CML resistant cells, indicating that 3-AT can enhance the sensitivity of this type of resistant cells to multiple TKIs and has a broad-spectrum sensitizing effect.

[0013] (3) This invention provides direct evidence that 3-AT is an effective drug for treating BCR::ABL amplification / overexpression type CML resistance, and has high clinical translational value. Attached Figure Description

[0014] Figure 1 CML cells showing BCR::ABL amplification / overexpression are resistant to TKIs;

[0015] Figure 2 The results showed that CAT activity was higher in CML cells with BCR::ABL amplification / overexpression, and 3-AT could effectively inhibit CAT activity;

[0016] Figure 3 The results showed that 3-AT can effectively inhibit CML cell proliferation;

[0017] Figure 4 The results showed that 3-AT could reduce the IC50 value of imatinib in CML cells;

[0018] Figure 5 The results showed that 3-AT could reduce the IC50 value of dasatinib in CML cells;

[0019] Figure 6 The results showed that 3-AT could reduce the IC50 value of nilotinib in CML cells;

[0020] Figure 7 The results showed that 3-AT could reduce the IC50 value of ponatinib in CML cells;

[0021] Figure 8The results showed that CAT activity was increased in bone marrow cells of CML patients, and 3-AT combined with imatinib synergistically inhibited the proliferation of CML bone marrow cells. Specific implementation methods

[0022] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0023] The specific experimental procedure will be given below.

[0024] 1. Detection of the effect of BCR::ABL amplification / overexpression on the sensitivity of cells to TKIs: Fluorescence in situ hybridization (FISH) and Western blot experiments were used to confirm the amplification / overexpression of the BCR::ABL fusion gene and protein in KCL22 and K562 / G01 cells. Then, the 48-hour IC50 values ​​of the cells to four TKIs were detected by CCK-8 assay.

[0025] 2. Detection of the effect of BCR::ABL amplification / overexpression on cellular CAT activity: cellular CAT expression and activity were detected by Western blot assay and a catalase (CAT) activity assay kit (ammonium molybdate colorimetric method).

[0026] 3. Effects of 3-AT on CAT enzyme activity and cell proliferation: Changes in CAT activity in K562, KCL22, and K562 / G01 cells before and after 3-AT treatment were detected using a CAT activity assay kit (ammonium molybdate colorimetric method), confirming that 3-AT can effectively inhibit CAT enzyme activity in cells. The inhibitory effect of 3-AT on cell proliferation at 72 hours was detected by CCK-8 assay to clarify the time-effect relationship of 3-AT on cell proliferation inhibition, and the IC50 value of cells to 3-AT at 48 hours was further evaluated.

[0027] 4. Effect of 3-AT on the sensitivity of BCR::ABL amplified / overexpressed cells to TKIs: The IC50 values ​​of cells with and without CAT at 48 hours were detected by CCK-8 assay.

[0028] 5. Detection of the combined effect of 3-AT and imatinib in primary cells of CML patients: Bone marrow samples were collected from CML patients, and bone marrow mononuclear cells were isolated. A 4×4 concentration matrix of 3-AT and imatinib was set up. After 48 h of combined treatment, cell viability was detected by CCK-8 assay. The combined effect of the two drugs was evaluated using a ZIP model, and the ZIP index was calculated to quantify the degree of synergistic effect.

[0029] 6. Experimental Methods:

[0030] (1) The method for culturing leukemia cells and the method for dissolving the drugs used in the embodiments of the present invention:

[0031] The CML cell lines K562, KCL22, and K562 / G01 used in this invention were routinely cultured and passaged in RPMI-1640 complete medium containing 10% fetal bovine serum at 37 ℃ in a 5% CO2 incubator. Cell status was observed daily, and the medium was changed and passaged regularly. Cells in the logarithmic growth phase were used for subsequent experiments. Drug preparation methods are as follows: TKIs (imatinib, dasatinib, nilotinib, ponatinib) were prepared with DMSO as the solvent for storage; 3-AT was dissolved and stored in sterile PBS.

[0032] (2) The method for isolating bone marrow and peripheral blood mononuclear cells in the embodiments of the present invention:

[0033] Bone marrow samples from CML patients and peripheral blood samples from healthy individuals were collected. Mononuclear cells were isolated using the Ficoll-Hypaque density gradient centrifugation method. An equal volume of Ficoll-Hypaque separation buffer (density 1.077 g / mL) was slowly added to the bone marrow or peripheral blood sample, and the mixture was centrifuged at 15,000 rpm for 30 min at 4 °C. After centrifugation, mononuclear cells from the middle white membrane layer were aspirated and washed twice with sterile PBS, centrifuged at 15,000 rpm for 15 min each time. Finally, 1 mL of erythrocyte lysis buffer was added, and the erythrocytes were lysed on ice for 5 min. After lysis, the cells were centrifuged at 15,000 rpm for 5 min. The isolated mononuclear cells were used for subsequent Western blot analysis, CAT enzyme activity assays, and in vitro drug sensitivity experiments.

[0034] (3) The method for detecting protein expression levels by Western blotting in the embodiments of the present invention:

[0035] 1) Extraction and quantification of total cellular protein: Collect cells, wash three times with pre-cooled PBS, remove the PBS, add the appropriate volume of premixed lysis buffer according to the amount of cell pellet, vortex thoroughly, and then sonicate. After complete lysis, centrifuge at 4℃ and 13000 rpm for 30 min. Transfer the supernatant containing total protein to a new EP tube, mix well, and take 2 μl to determine the protein concentration using the BCA method, recording the remaining volume of the supernatant. Add 1 / 4 volume of 5× loading buffer to the supernatant, mix well, and heat in a 100℃ metal bath for 10 min to fully denature the protein. After completion, store at -40℃ for later use.

[0036] 2) Electrophoretic separation: Prepare a separating gel of appropriate concentration according to the molecular weight of the target protein. After pouring the gel, add 1 ml of anhydrous ethanol for sealing. Let it stand at room temperature for 30 min until the gel solidifies. Remove the ethanol and blot the remaining liquid with filter paper. Insert the comb to prepare a 5% stacking gel and slowly add it to the top of the separating gel. Let it stand at room temperature for 30 min. Calculate the loading amount based on the protein quantification results. Use a two-step electrophoresis method: Step 1: 80 V, 100 mA electrophoresis for 30 min; Step 2: 120 V, 100 mA electrophoresis for 90 min.

[0037] 3) Protein Transfer: Cut a PVDF membrane to an appropriate size according to the molecular weight of the target protein, activate it in methanol for 30 seconds, rinse with distilled water for 2 minutes, and equilibrate the PVDF membrane and filter paper in pre-cooled wet transfer buffer. Cut a piece of gel containing the target protein region and equilibrate it in the same wet transfer buffer. Stack the sponge, filter paper, PVDF membrane, gel, filter paper, and sponge in the order from positive to negative electrode. Transfer the membrane at a constant current of 220 mA, adjusting the transfer time according to the molecular weight of the protein.

[0038] 4) Sealing: After the transfer is completed, the PVDF membrane is placed in a sealing solution containing 5% skim milk powder and sealed on a shaker at room temperature for 2 hours.

[0039] 5) Antigen-antibody reaction: Place the blocked PVDF membrane on a wax plate, add the primary antibody working solution diluted to the optimized ratio, ensuring complete coverage of the membrane surface, and incubate overnight at 4 °C. After recovering the primary antibody, wash the membrane three times with TBST for 8 min each time. Then add the HRP-labeled secondary antibody diluted to the optimized ratio, incubate at room temperature for 2 h, and wash the membrane three times with TBST for 8 min each time.

[0040] 6) Chemiluminescence detection: Place the PVDF film on a wax plate, add freshly prepared chemiluminescence substrate solution (solution A:solution B = 1:1), cover the film surface, and then expose and develop it in the imaging system.

[0041] (4) Specific methods for detecting the cell proliferation inhibition rate after single and combined treatment with TKI or CAT inhibitors using the Cell Counting Kit-8 (CCK-8) in this invention example:

[0042] K562, KCL22, K562 / G01 and primary bone marrow mononuclear cells in logarithmic growth phase were collected and seeded in 96-well plates at a density of 4000 cells per well, and different concentrations of drugs were added.

[0043] 1) Single-drug treatment: Different concentrations of imatinib (0, 0.01, 0.1, 1, 10, 20 μM), nilotinib (0, 0.001, 0.01, 0.1, 1, 10 μM), dasatinib (0, 0.001, 0.01, 0.1, 1, 10 μM), ponatinib (0, 0.001, 0.01, 0.1, 1, 10 μM), or 3-AT (0, 0.001, 0.005, 0.01, 0.1, 0.5 M) were added to each treatment group, with 3 replicates for each group.

[0044] 2) Combined treatment: 3-AT at a final concentration of 20 mM was added to each concentration gradient of imatinib, nilotinib, dasatinib, or ponatinib to evaluate the effect of CAT inhibitors on TKI sensitivity. Each treatment group had three replicates.

[0045] 3) Synergistic effect of drugs: In primary mononuclear cells from the bone marrow of CML patients, different concentrations of imatinib combined with different concentrations of 3-AT were set up. The concentration gradients of imatinib were set at 0, 0.5, 1, and 10 μM, and the concentration gradients of 3-AT were set at 0, 2, 5, and 10 mM. Each concentration combination was added according to a matrix design (4×4), and each treatment group had 3 replicates.

[0046] Add RPMI-1640 complete medium containing 10% FBS to each well to a final volume of 100 μl. Incubate at 37 ℃ in a 5% CO2 incubator. After 48 h, add 10 μl of CCK-8 reagent to each well and incubate at 37 ℃ in the dark for 2 h. Detect the absorbance at 450 nm using a microplate reader.

[0047] To establish a baseline and provide a direct comparison of the effects of 3-AT intervention on the sensitivity of CML cells to TKIs, this study used the mean IC50 value of the control group treated with TKIs alone as a benchmark and normalized it to 1. The measured IC50 values ​​of each 3-AT combined with TKI treatment group were divided by the mean IC50 value of the control group to calculate the relative IC50 value of each group. This relative IC50 value was used to quantitatively evaluate the changes in cell sensitivity to TKIs before and after CAT inhibitor intervention. A relative IC50 value less than 1 indicates increased drug sensitivity, while a relative IC50 value greater than 1 indicates decreased drug sensitivity.

[0048] (5) Specific method for detecting CAT enzyme activity in the examples of this invention:

[0049] 1) Basic CAT activity assay: K562, KCL22, K562 / G01 cells and primary bone marrow mononuclear cells in logarithmic growth phase were collected, counted, and 5×10⁻⁶ cells were taken. 6 Cells were washed twice with pre-chilled PBS, and the cell pellet was resuspended in 1 mL of extraction buffer according to the kit instructions. Lysis was performed on ice for 30 min, with shaking every 10 min. After lysis, the cells were centrifuged at 12000 rpm for 15 min at 4 ℃, and the supernatant was used for CAT enzyme activity assay. Then, working solution of Reagent II was prepared according to the experimental dosage at a ratio of 1:99 (Reagent II:Reagent I). Two 1.5 mL EP tubes were taken from each sample, divided into a control group and an assay group. The control group was added with 100 μL ddH2O and 100 μL of Reagent II working solution, while the assay group was added with 100 μL crude enzyme solution and 100 μL of Reagent II working solution. After mixing, the mixture was incubated in a 25 ℃ metal bath for 2 min. Then, 300 μL of Reagent III and 800 μL of Reagent IV were added sequentially to the two EP tubes, mixed, and 300 μL was added to a 96-well plate. The absorbance was measured at 405 nm using a microplate reader.

[0050] 2) CAT activity assay after 3-AT treatment: K562, KCL22, K562 / G01 cells and primary bone marrow mononuclear cells in logarithmic growth phase were collected, counted, and 1×10⁻⁶ cells were taken. 7 Cells were seeded into 6-well plates, and 0 mM (control group) and 20 mM 3-AT were added to each well. RPMI-1640 complete medium was added to a final volume of 2 ml per well, and the plates were incubated at 37 ℃ in a 5% CO2 incubator for 24 h. After incubation, cells from each group were collected, washed twice with pre-chilled PBS, and the cell pellet was resuspended in an appropriate amount of extraction buffer. The cells were lysed on ice for 30 min, centrifuged at 12000 rpm for 15 min at 4 ℃, and the supernatant was collected for CAT detection according to the above steps.

[0051] (6) Specific method for detecting BCR::ABL fusion gene by FISH during the intermediate phase in this invention:

[0052] Collect K562, KCL22, and K562 / G01 cells in logarithmic growth phase, wash twice with pre-chilled PBS, then add 7 mL of pre-warmed 0.075 M KCl hypotonic buffer, mix by pipetting, and incubate at 37 ℃ for 30 min. Then, add 1.5 mL of freshly prepared Carnoy's fixative (methanol:glacial acetic acid = 3:1) dropwise, mix gently, pre-fix at 37 ℃ for 5 min, centrifuge at 300 g for 5 min, and discard the supernatant. Add 7 mL of Carnoy's fixative again, gently disperse the cells, fix at room temperature for 5 min, centrifuge at 300 g for 5 min, and discard the supernatant. Repeat fixation three times until cell clumps are clear and the supernatant is clear. Resuspend the cell pellet in 500 μL of fixative, disperse into a light mist, and drop 2-3 drops from a height of 15 cm onto a glass slide, immediately disperse, and air dry at room temperature. Age the slide in a 75 ℃ oven for 30 min. After aging, the slides were sequentially dehydrated in 70%, 85%, and 100% ethanol gradients for 2 min each, and then air-dried. The BCR::ABL dual-color dual-fusion probe was added to the target area of ​​the slide, and a coverslip was placed on top to seal the edges. The slide was placed in a hybridization apparatus and denatured at 85 °C for 5 min, followed by hybridization at 37 °C overnight. After hybridization, the coverslip was removed, and the slide was placed in 0.4×SSC / 0.3% NP-40 solution and washed with shaking in a 73 °C water bath for 2 min; then placed in 2×SSC / 0.1% NP-40 solution and washed at room temperature for 1 min. The slides were then air-dried and mounted with DAPI counterstain. The results were observed using a fluorescence microscope.

[0053] 6. Experimental Results:

[0054] (1) Cells with BCR::ABL amplification / overexpression are resistant to TKIs

[0055] Figure 1 A shows the results of FISH detection of the BCR::ABL fusion gene in K562, KCL22, and K562 / G01 cells. K562 and K562 / G01 cells showed a single fusion signal, while KCL22 cells showed two fusion signals, with the fusion signal intensity being stronger in K562 / G01 cells, suggesting that the BCR::ABL fusion gene is amplified / overexpressed in KCL22 and K562 / G01 cells. Figure 1B represents the results of Western blot analysis of BCR::ABL protein expression and phosphorylation levels in the three cell lines. Compared with K562 cells, the total BCR::ABL protein and p-BCR::ABL (Tyr245) phosphorylation levels were significantly increased in KCL22 and K562 / G01 cells, suggesting enhanced BCR::ABL kinase activity in both cell lines. Figure 1 C represents the IC50 values ​​of the three cell lines against four TKIs as determined by the CCK-8 assay. The IC50 values ​​of KCL22 and K562 / G01 cells against some TKIs were significantly higher than those against K562 cells, indicating that BCR::ABL amplification / overexpression cells exhibit resistance to multiple TKIs. These results confirm the presence of BCR::ABL fusion gene amplification and protein overexpression in KCL22 and K562 / G01 cells, and their higher resistance to TKIs, suggesting that BCR::ABL amplification / overexpression is a crucial driving factor for TKI resistance in CML cells.

[0056] (2) Upregulation of CAT expression and activity in CML cells with BCR::ABL amplification / overexpression

[0057] Figure 2 A shows the results of Western blot analysis of CAT protein expression in K562, KCL22, and K562 / G01 cells. Compared with K562 cells, the expression levels of CAT protein in KCL22 and K562 / G01 cells were significantly increased. Figure 2 B shows the results of CAT activity assays in the three cell lines. The CAT activity in KCL22 and K562 / G01 cells was significantly higher than that in K562 cells, consistent with the trend of protein expression level changes. Figure 2 C represents the changes in CAT enzyme activity before and after 3-AT treatment. After 24 h of 3-AT treatment, CAT enzyme activity in CML cells was significantly reduced, indicating that 3-AT can effectively inhibit CAT enzyme activity in cells. These results confirm that CAT expression and enzyme activity are significantly increased in BCR::ABL amplified / overexpressing cells, and that 3-AT can effectively inhibit CAT activity.

[0058] (3) 3-AT can effectively inhibit the proliferation of CML cells.

[0059] Figure 3 A shows the proliferation curve of K562 cells treated with 3-AT for 72 h, as detected by the CCK-8 assay. Figure 3 B is the proliferation curve of K562 cells after 72 h of 3-AT treatment, as detected by the CCK-8 assay. Figure 3C represents the proliferation curve of K562 cells treated with 3-AT for 72 h, as detected by the CCK-8 assay. The results showed that 3-AT significantly inhibited the proliferation of the three cell lines, and the degree of inhibition was time-dependent. Figure 3 D represents the results of CCK-8 assay detection of the inhibitory effects of different concentrations of 3-AT on the proliferation of the three cell lines. The IC50 values ​​of 3-AT in KCL22 and K562 / G01 cells were significantly higher than those in K562 cells, suggesting that BCR::ABL amplified / overexpressing cells require higher concentrations of 3-AT to achieve the same inhibitory effect on proliferation due to enhanced CAT activity. These results indicate that 3-AT can effectively inhibit CML cell proliferation, while the sensitivity of BCR::ABL amplified / overexpressing cells to 3-AT is significantly reduced, further confirming the functional importance of CAT in BCR::ABL amplified / overexpressing cells.

[0060] (4) The CAT inhibitor 3-AT can significantly enhance the sensitivity of BCR::ABL amplified / overexpressing CML cells to imatinib.

[0061] Figure 4 The effect of 3-AT combined with imatinib treatment on the IC50 values ​​of K562 cells (A), KCL22 cells (B), and K562 / G01 cells (C) was detected by CCK-8 assay. The results showed that the IC50 values ​​of imatinib in all three cell lines were significantly reduced after the combination of 3-AT and imatinib treatment compared with the imatinib monotherapy group.

[0062] (5) The CAT inhibitor 3-AT can significantly enhance the sensitivity of BCR::ABL amplified / overexpressing CML cells to dasatinib.

[0063] Figure 5 The effect of 3-AT combined with dasatinib treatment on the IC50 values ​​of K562 cells (A), KCL22 cells (B), and K562 / G01 cells (C) was detected using the CCK-8 assay. The results showed that the IC50 values ​​of dasatinib in all three cell lines were significantly reduced after the combination of 3-AT and dasatinib treatment compared with the dasatinib monotherapy group.

[0064] (6) The CAT inhibitor 3-AT can significantly enhance the sensitivity of BCR::ABL amplified / overexpressing CML cells to nilotinib.

[0065] Figure 6The effect of 3-AT combined with nilotinib treatment on the IC50 values ​​of K562 cells (A), KCL22 cells (B), and K562 / G01 cells (C) was detected by CCK-8 assay. The results showed that the IC50 values ​​of nilotinib in all three cell lines were significantly reduced after the combination with 3-AT compared with the nilotinib monotherapy group.

[0066] (7) The CAT inhibitor 3-AT can significantly enhance the sensitivity of BCR::ABL amplified / overexpressing CML cells to ponatinib.

[0067] Figure 7 The effect of 3-AT combined with ponatinib treatment on the IC50 values ​​of K562 cells (A), KCL22 cells (B), and K562 / G01 cells (C) was detected by CCK-8 assay. The results showed that the IC50 values ​​of ponatinib in all three cell lines were significantly reduced after the combination of 3-AT and ponatinib treatment compared with the ponatinib monotherapy group.

[0068] (8) The CAT inhibitor 3-AT can effectively inhibit CAT activity and enhance imatinib sensitivity in primary cells of CML patients.

[0069] Figure 8 A shows the results of Western blot analysis of CAT protein expression in primary bone marrow mononuclear cells from CML patients and primary peripheral blood mononuclear cells from healthy controls. Compared with healthy controls, the expression level of CAT protein in bone marrow mononuclear cells from CML patients was significantly increased. Figure 8 B shows the changes in CAT enzyme activity after 3-AT treatment of primary bone marrow mononuclear cells from CML patients. Treatment with 3-AT significantly reduced CAT enzyme activity in bone marrow mononuclear cells from CML patients, indicating that 3-AT can effectively inhibit CAT activity in primary cells from CML patients. Figure 8 C represents the results of CCK-8 assay on the inhibition of bone marrow mononuclear cell proliferation in CML patients by imatinib monotherapy and in combination with 3-AT. Compared with the imatinib monotherapy group, the IC50 value of imatinib was significantly reduced after combination with 3-AT. Figure 8To further evaluate the combined effect of 3-AT and imatinib in primary cells of CML patients using the ZIP (Zero Interaction Potency) model, the results showed a ZIP synergistic score of 22.214, indicating a significant synergistic effect. These results were further confirmed in clinical patient specimens that CAT expression and activity were significantly elevated in CML patients, and the CAT inhibitor 3-AT effectively inhibited CAT activity and enhanced TKI sensitivity, while also exhibiting a synergistic effect with imatinib. This suggests that targeting CAT in combination with TKIs has promising application prospects in the clinical treatment of CML.

[0070] The embodiments described in this invention are merely preferred implementations of this invention and are not intended to limit this invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. 3-AT in the treatment of TKI resistance in chronic myeloid leukemia caused by BCR::ABL amplification / overexpression.

2. The application as described in claim 1, characterized in that, The 3-AT is 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase (CAT).

3. The application as described in claim 1, characterized in that, The BCR::ABL amplification / overexpression chronic myeloid leukemia (CML) includes CML with BCR::ABL fusion gene amplification and BCR::ABL fusion protein overexpression; the drug resistance refers to the resistance of BCR::ABL amplification / overexpression CML cells to tyrosine kinase inhibitors (TKIs); the TKIs include imatinib, nilotinib, dasatinib, and ponatinib.

4. The application as described in claim 1, characterized in that, The 3-AT enhances the sensitivity of BCR::ABL-amplified / overexpressing CML cells to TKIs by inhibiting CAT activity.

5. The application as described in any one of claims 1-4, characterized in that, The 3-AT plays the following role in CML amplified / overexpressed by BCR::ABL: (1) Inhibits CAT enzyme activity; (2) Inhibits cell proliferation; (3) Enhance the sensitivity of cells to TKIs.

6. The application or pharmaceutical composition according to any one of claims 1-5, characterized in that, The 3-AT overcomes CML TKI resistance caused by BCR::ABL amplification / overexpression by inhibiting CAT.

Images