Use of small molecule inhibitor pz1 in the treatment of kit mutation-positive acute myeloid leukemia

Abstract

The application discloses application of a small molecule inhibitor PZ1 in treating KIT mutation positive acute myeloid leukemia. Researches of the application show that PZ1 can significantly inhibit the proliferation of a plurality of KIT mutation positive leukemia cells and promote cell apoptosis at a cell level, and the effect is significantly better than that of an existing TKI drug; further researches show that PZ1 can stably combine with KIT protein and inhibit the phosphorylation level of KIT and downstream STAT5, AKT, ERK and other signal paths. In-vivo research results show that PZ1 can reduce the proportion of leukemia cells in peripheral blood of mice, significantly reduce the infiltration of leukemia cells in bone marrow and spleen, and reduce the weight of spleen. Therefore, the application discloses the treatment effect of PZ1 in KIT mutation positive leukemia, and can effectively overcome the TKI drug resistance of the existing KIT mutation positive leukemia, solve the treatment difficulty of the current KIT mutation positive leukemia drug resistance, and provide a new strategy for the treatment of the disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the small molecule inhibitor PZ1 in the treatment of KIT mutation-positive acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a disease characterized by abnormal proliferation, differentiation arrest, and impaired apoptosis of hematopoietic stem cells and their precursor cells during differentiation. This results in a large number of myeloid blast cells, inhibiting normal hematopoietic function and infiltrating other tissues and organs. While treatment of AML has significantly improved thanks to traditional chemotherapy combined with allogeneic hematopoietic stem cell transplantation, overall survival remains unsatisfactory. The 5-year relapse-free survival rate is less than 50% for young adult patients and less than 12% for elderly patients. The efficacy of chemotherapy in AML patients is closely related to whether they carry gene mutations. More than 50% of AML patients carry receptor tyrosine kinase mutations, such as KIT and FLT3 mutations. These mutations are important factors contributing to poor response to conventional chemotherapy, high relapse rates, and short overall survival. In AML, the overall incidence of KIT mutations is approximately 4%-14%, particularly prevalent in core-binding factor leukemia (CBF-AML) subtypes such as t(8;21), inv(16), or t(16;16), accounting for about 25%-40%. Common mutation types primarily occur in exon 8, which encodes the extracellular domain, and in exon 17, which encodes the kinase domain. Among these, D816V and N822K mutations occurring in the activation loop of the kinase domain are the most common. These mutations lead to persistent KIT activation and cause abnormal activation of multiple downstream intracellular signaling pathways, including STAT5, PI3K, and MAPK, thereby promoting excessive cell proliferation or inhibiting apoptosis. KIT mutations (especially D816V) are a poor prognostic indicator, particularly in AML1-ETO fusion gene-positive patients, where mutations are closely associated with acute transformation, relapse, and poor prognosis in leukemia. Therefore, targeting KIT mutations is an effective treatment for KIT-mutant leukemia.

[0003] Currently, the main targeted therapies for KIT mutations are tyrosine kinase inhibitors (TKIs). The application of TKIs has greatly improved the clinical treatment of KIT-mutant AML patients, but most patients develop resistance within months or a year after treatment, leading to disease relapse or progression. Imatinib was the first approved first-generation TKI for the treatment of KIT-mutant gastric stromal tumors. It also showed some efficacy against KIT wild-type or partially extracellular KIT-mutant AML, but it was ineffective against mutations occurring in the kinase domain activation loop, especially the D816V mutation, and resistance developed during treatment due to secondary mutations in the kinase domain. Second-generation TKIs can inhibit multiple tyrosine kinase receptors such as KIT, PDGFR, and VEGFR; representative drugs include sunitinib and dasatinib. Second-generation TKIs have stronger inhibitory effects on some imatinib-resistant KIT mutations. However, their inhibitory effect on the KIT D816V mutation is not ideal. Third-generation TKIs have stronger inhibitory activity; representative drugs include midostaurin and avatinib. Midotuximab and avatinib were approved by the FDA in 2017 and 2021, respectively, for the treatment of advanced systemic mastocytosis. Avatinib is also approved for the treatment of unresectable or metastatic gastrointestinal stromal tumors harboring KIT or PDGFRA mutations. Several clinical studies are currently underway for avatinib in KIT-mutant AML patients, demonstrating promising therapeutic prospects. However, a retrospective study analyzing 20 patients with t(8;21) AML and KIT mutations who responded poorly to immunotherapy after allogeneic hematopoietic stem cell transplantation but received avatinib treatment, with a median follow-up of 5.5 months (2.0–10.0 months), only 45% of patients achieved negative RUNX1-RUNX1T1 fusion gene conversion, and 3 patients achieved hematologic remission and survived. This illustrates the relapsed / refractory nature of avatinib treatment with KIT mutations, indicating that some patients with t(8;21) AML still do not benefit from it. Therefore, there is an urgent need to develop new inhibitors that can overcome resistance to TKIs in KIT-mutant positive AML.

[0004] PZ1 (CAS: 1800505-64-9) is a novel VEGFR2 and RET tyrosine kinase inhibitor that blocks the blood supply required for RET-stimulated growth, thereby inhibiting tumor growth. In animal studies, PZ1 showed no significant toxicity at 100.0 mg / kg, suggesting a large therapeutic window. However, whether PZ1's inhibitory effect on KIT-mutant AML is superior to existing TKIs and whether it can overcome TKI resistance has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of the small molecule inhibitor PZ1 in the preparation of a drug for treating KIT mutation-positive acute myeloid leukemia.

[0006] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0007] KIT mutation is an important molecular marker of relapse and poor prognosis in acute myeloid leukemia (AML), especially in patients with AML1-ETO fusion gene positivity, where it is closely associated with acute transformation, relapse, and poor prognosis. While tyrosine kinase inhibitors have improved patient outcomes, drug resistance remains a significant issue. This invention, using CellTiter-Glo experiments, reveals that the small molecule inhibitor PZ1 significantly inhibits the proliferation of various KIT-mutant positive leukemia cell lines (Ba / F3 KIT-mutant (D816V, N822K, D816F, D816H, D816Y, V560D, V560G) and Kasumi-1 cells at the cellular level. Flow cytometry analysis further shows that PZ1 significantly promotes leukemia cell apoptosis, with effects significantly superior to existing TKI drugs (avatinib, dasatinib, and imatinib), indicating that PZ1 has a broad-spectrum inhibitory effect on KIT mutations and a stronger inhibitory effect. Furthermore, Western blotting experiments revealed that PZ1 stably binds to the KIT protein and inhibits the phosphorylation levels of KIT and its downstream signaling pathways such as STAT5, AKT, and ERK. This is the first publicly disclosed evidence that PZ1, as a VEGFR2 and RET tyrosine kinase inhibitor, can inhibit the abnormal activation of the KIT signaling pathway. In vivo experiments showed that, using a PDX mouse model constructed from bone marrow cells of leukemia patients resistant to avatinib, PZ1 effectively inhibited the proliferation of leukemia cells in peripheral blood and reduced the infiltration of leukemia cells in the bone marrow and spleen. The results of this invention fully demonstrate that the small molecule inhibitor PZ1 can effectively inhibit the proliferation of KIT-mutant leukemia cells and promote their apoptosis. Its inhibitory effect is significantly better than existing TKI drugs, and it can effectively overcome TKI resistance in KIT-mutant positive leukemia, solving the current treatment challenges of drug resistance in KIT-mutant positive leukemia and providing a new treatment strategy for this disease.

[0008] Therefore, the present invention provides the use of the small molecule inhibitor PZ1 in the preparation of a drug for treating KIT mutation-positive acute myeloid leukemia, wherein the structural formula of the small molecule inhibitor PZ1 is shown in the following formula (Ⅰ):

[0009] .

[0010] Furthermore, the KIT mutation-positive acute myeloid leukemia is drug-resistant KIT mutation-positive acute myeloid leukemia.

[0011] Furthermore, the drug resistance is resistance to avatinib, dasatinib, or imatinib. That is, the present invention provides the use of the small molecule inhibitor PZ1 in the preparation of a medicament for treating KIT mutation-positive acute myeloid leukemia resistant to avatinib, dasatinib, or imatinib.

[0012] Furthermore, the KIT mutation is one or more of the following KIT mutations: D816V, N822K, D816F, D816H, D816Y, V560D, and V560G.

[0013] Furthermore, the drug achieves its therapeutic effect by inhibiting the proliferation of leukemia cells and promoting their apoptosis.

[0014] Furthermore, the drug achieves its therapeutic effect by inhibiting the infiltration of leukemia cells in the bone marrow and spleen.

[0015] Furthermore, the drug achieves therapeutic effects by inhibiting the phosphorylation levels of KIT and its downstream STAT5, AKT, or ERK signaling pathways.

[0016] Furthermore, the concentration of the small molecule inhibitor PZ1 in the drug is 1–100 nM.

[0017] Furthermore, the solvent for the small molecule inhibitor PZ1 in the drug is DMSO, Tween 20, xanthan gum, and water.

[0018] Furthermore, the solvent consists of 5% DMSO, 1% Tween 20, and 0.125% xanthan gum; the 5% DMSO, 1% Tween 20, and 0.125% xanthan gum are diluted with water.

[0019] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0020] Furthermore, the dosage form of the drug is oral or injectable.

[0021] This invention, based on the known pharmacology of PZ1, demonstrates through extensive in vitro cell experiments, flow cytometry, clinical samples, and in vivo animal model experiments that PZ1 can significantly inhibit the proliferation of KIT-mutant leukemia cells, promote their apoptosis, and significantly inhibit the proportion of leukemia cells in mouse bone marrow and peripheral blood, and reduce their infiltration in bone marrow and spleen. Its inhibitory effect is significantly superior to existing TKI drugs. Compared to existing TKI drugs, the PZ1 small molecule inhibitor has a broader and stronger inhibitory effect on KIT mutations and can effectively overcome TKI resistance. This invention overcomes the shortcomings of existing technologies, reveals the therapeutic effect of PZ1 in KIT-mutant positive AML, and discovers that PZ1 can overcome existing TKI resistance in KIT-mutant positive AML and can be applied to scientific research and clinical treatment.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides the application of the small molecule inhibitor PZ1 in the treatment of KIT mutation-positive acute myeloid leukemia. In vitro studies show that PZ1 significantly inhibits the proliferation of various KIT mutation-positive leukemia cells and Kasumi-1 cells at the cellular level, promotes apoptosis of leukemia cells, and exhibits significantly better effects than existing TKI drugs, indicating that PZ1 has a broad-spectrum inhibitory effect on KIT mutations and a stronger inhibitory effect. Further studies show that PZ1 can stably bind to KIT protein and inhibit the phosphorylation levels of KIT and its downstream signaling pathways such as STAT5, AKT, and ERK. In vivo studies show that PZ1 can significantly reduce the proportion of leukemia cells in mouse peripheral blood, significantly reduce the infiltration of leukemia cells in the bone marrow and spleen, and reduce the spleen weight in mice. Therefore, this invention reveals the therapeutic effect of PZ1 in KIT mutation-positive leukemia and can effectively overcome the problem of existing TKI resistance in KIT mutation-positive leukemia, solving the treatment difficulties of drug resistance in KIT mutation-positive leukemia, and can be further applied to scientific research and clinical treatment, providing a new treatment strategy for this disease. Attached Figure Description

[0024] Figure 1 To investigate the effects of treating cell lines containing KIT resistance mutations, as well as P815 and Kasumi-1 cells, with the same concentration gradients of PZ1, avatinib, dasatinib, and imatinib on cell viability.

[0025] Figure 2 To investigate the effects of treating Ba / F3 KIT D816V mutant or Ba / F3 KIT N822K mutant cell lines with the same concentration gradient of PZ1, avatinib, and dasatinib on apoptosis.

[0026] Figure 3PZ1 binds to the KIT protein and inhibits the KIT pathway and its downstream STAT5, AKT, and ERK signaling pathways. Figure 3 In the diagram, AB represents the cell thermodynamic migration assay used to detect the binding of PZ1 to KIT protein; CG represents the Western blot assay used to detect the effect of PZ1 on the phosphorylation of KIT and its downstream STAT5, AKT and ERK.

[0027] Figure 4 The study investigated the significant inhibition of leukemia cell proliferation and tissue infiltration by PZ1 in a KIT-mutant positive PDX mouse model of leukemia. Figure 4 In the diagram, A represents the construction and experimental process of the PDX mouse model; BE represents the proportion of leukemia cells in mouse peripheral blood and the infiltration of leukemia cells in bone marrow and spleen detected by flow cytometry; and F represents the photographic results of mouse spleen weight and size. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] PZ1 (CAS: 1800505-64-9) has the following structural formula:

[0031] .

[0032] Dissolution and dilution of PZ1 small molecule inhibitors: Dissolution methods include in vitro and in vivo applications. For in vitro application, PZ1 is dissolved in DMSO to obtain a 10 mM stock solution, which is stored at -20°C. Before use, the stock solution is diluted with DMSO to the required concentration (1-100 nM). For in vivo application, PZ1 is dissolved in 5% DMSO + 1% Tween 20 + 0.125% xanthan gum + ddH2O, and administered to mice by gavage at a dose of 60 mg / kg.

[0033] Example 1: Effects of PZ1 on the viability and apoptosis of KIT mutant positive cell lines

[0034] I. Experimental Methods

[0035] 1. CellTiter-Glo assay for cell viability

[0036] (1) Constructing multiple leukemia cell lines carrying KIT mutations. KIT mutant plasmids (D816V, N822K, D816F, D816H, D816Y, V560D, V560G) were overexpressed in Ba / F3 cells by lentiviral infection to construct the corresponding mutant lines.

[0037] (2) Ba / F3 KIT mutant (D816V, N822K, D816F, D816H, D816Y, V560D, V560G) cell lines, as well as P815 and Kasumi-1 cells, were cultured in RPMI-1640 medium containing 10% serum, 1% penicillin and streptomycin and placed in a constant temperature incubator at 37℃ and 5% CO2 to screen out stable overexpression lines.

[0038] (3) Use the solvent DMSO to prepare a 10 mM stock solution of compounds PZ1, avapritinib, dasatinib and imatinib and store it at -20℃.

[0039] (4) Seed the cells in good growth condition into 96-well plates and treat them with the same concentration gradient of PZ1, avatinib, dasatinib and imatinib for 48 hours. Remove the cell plate and add 100 µL CellTiter-Glo reagent to each well. Incubate at room temperature in the dark for 30 minutes. After gently mixing by pipetting, aspirate 60 µL from each well into a 384-well plate and read the fluorescence value using a microplate reader. Calculate the cell viability based on the fluorescence value analysis.

[0040] 2. Flow cytometry detection of apoptosis

[0041] (1) Ba / F3 KIT D816V mutant or Ba / F3 KIT N822K mutant cell lines were treated with the same concentrations of PZ1, avatinib, and dasatinib for 48 hours. Cell samples were collected, washed once with pre-cooled PBS, and then washed once with 1× binding buffer. The cells were then centrifuged at 400g and 4℃ for 5 minutes, and the supernatant was discarded. The cells were resuspended in 100 µL of 1× binding buffer, and 5 µL of Annexin V-APC and 2 µL of PI were added. The mixture was gently mixed and incubated at room temperature in the dark for 15 minutes. After adding 200 µL of 1× binding buffer, cell apoptosis was detected by flow cytometry.

[0042] II. Experimental Results

[0043] Cell viability test results as follows Figure 1As shown, when cell lines containing KIT resistance mutations (D816V or N822K) and P815 and Kasumi-1 cells were treated with the same concentration gradient, PZ1 showed significantly stronger inhibitory effects on KIT resistance mutant cell lines and P815 and Kasumi-1 cells than avatinib, dasatinib, and imatinib. PZ1 also inhibited cell lines with other mutations at other sites of Ba / F3 KIT D816 (D816F, D816H, D816Y) and mutations at the V560 site (V560D, V560G), and its inhibitory effect was significantly better than that of avatinib and dasatinib. Figure 1 (Chinese AI); from Figure 1 As shown in Figure J, PZ1 showed significantly better inhibitory effects on KIT-resistant mutant cell lines than avatinib, dasatinib, and imatinib.

[0044] The results of the apoptosis experiment are as follows Figure 2 As shown, when Ba / F3 KIT D816V or Ba / F3 KIT N822K mutant cell lines were treated with the same concentration gradient of PZ1, avatinib, or dasatinib for 48 hours, PZ1 promoted apoptosis in a concentration-dependent manner, and the effect was significantly better than that of avatinib or dasatinib. This result indicates that the small molecule inhibitor PZ1 has a broader spectrum and stronger inhibitory effect on KIT-resistant mutant cell lines.

[0045] Example 2: PZ1 binds to KIT protein and inhibits the KIT pathway and its downstream STAT5, AKT and ERK signaling pathways.

[0046] I. Experimental Methods

[0047] 1. Cellular thermal shift assay (CESTA) to detect the binding of PZ1 to KIT protein.

[0048] Ba / F3 KIT N822K cells were treated with 1 μM PZ1 or an equal volume of DMSO (1 × 10⁻⁶ cells per cell). 7After 1 hour, cell samples were collected, washed with pre-cooled PBS, and resuspended in PBS buffer containing 1× protease inhibitor. The cells were then aliquoted into multiple reaction tubes. Cell samples were heated for 3 minutes at a temperature gradient of 37℃–50℃, cooled to room temperature, and then lysed three times using liquid nitrogen. The lysate was centrifuged at 12000 rpm at 4℃ for 15 minutes, and the supernatant was collected. An equal volume of 2× SDS was added, and the mixture was heated in a 100℃ metal bath for 10 minutes. KIT expression levels were then detected using Western blotting. Band grayscale values ​​were quantified using Image Lab software, and data analysis and plotting were performed using GraphPad Prism to evaluate the effect of PZ1 on KIT thermostability.

[0049] 2. Western blot assay to detect the effect of PZ1 on phosphorylation of KIT and its downstream STAT5, AKT and ERK.

[0050] (1) P815, Kasumi-1, Ba / F3 KIT D816V mutant cell line, Ba / F3 KIT N822K mutant cell line and PDX mouse bone marrow cells were cultured in RPMI-1640 medium containing 10% serum, 1% penicillin and streptomycin and placed in a constant temperature incubator at 37℃ and 5% CO2.

[0051] (2) Cells were treated with different concentration gradients of PZ1 (0-100 nM) for 6 hours, cell samples were collected, and the supernatant was discarded by centrifugation. Cells were lysed with an appropriate amount of protein lysis buffer and heated in a 100℃ metal bath for 10 minutes to obtain protein samples. Protein samples were placed in a 10% SDS-PAGE gel, with 30-50 μg of protein added to each well, and proteins of different molecular weights were separated by electrophoresis. The proteins were transferred to a PVDF membrane and blocked with 5% skim milk powder at room temperature for 1 hour. Then, the membrane was washed three times with 1×TBST buffer, and the prepared primary antibody (p-KIT, KIT, p-AKT, AKT, p-ERK, ERK, p-STAT5, STAT5, GAPDH) was added and incubated overnight at 4℃. Next, the membrane was washed three times with 1×TBST buffer for 10 minutes each time. The secondary antibody conjugated with horseradish peroxidase was diluted with 5% skim milk and incubated at room temperature for 1 hour. Wash three times with 1×TBST buffer, 10 minutes each time, and finally add developer to expose and develop in a developer apparatus.

[0052] II. Experimental Results

[0053] The results are as follows Figure 3 As shown, Figure 3The AB diagram results showed that the stability of KIT protein decreased with increasing temperature. However, after treatment with PZ1 in cells, the stability of KIT protein relatively increased with increasing temperature, indicating that PZ1 can promote the thermal stability of KIT protein. Figure 3 The CG plot results show that PZ1 inhibits the phosphorylation of KIT and its downstream AKT, ERK, and STAT5 in a concentration-dependent manner. This experimental result indicates that PZ1 can bind to and increase the stability of the KIT protein, and inhibit the aberrant activation of the KIT signaling pathway and its downstream signaling pathways.

[0054] Example 3: PZ1 significantly inhibited the proliferation and tissue infiltration of leukemia cells in a KIT mutant-positive leukemia PDX mouse model.

[0055] I. Experimental Methods

[0056] (1) Collect primary bone marrow cells from patients with KIT D816V-mutant (avatinib-resistant) leukemia who relapsed after avatinib treatment, and divide 1×10 6 Cells were injected into NOG mice via tail vein injection, and peripheral blood was collected from the mice periodically. The proportion of leukemia cells in the peripheral blood was detected by flow cytometry. When the proportion of leukemia cells was positive, the patient-derived xenograft (PDX) mouse model of KIT D816V mutant leukemia was successfully constructed.

[0057] (2) Mice were euthanized, and their bone marrow and spleen were collected to obtain bone marrow cells and spleen cells, which were then cryopreserved in liquid nitrogen. Some of the cells were injected into NOG mice via the tail vein. The mice were then randomly divided into 5 groups: control group, PZ1 group, avatinib group, dasatinib group, and imatinib group. After 14 days, the mice were administered equal volumes of solvent, 60 mg / kg PZ1, 30 mg / kg avatinib, 5 mg / kg dasatinib, and 100 mg / kg imatinib via gavage, respectively. Peripheral blood was collected from each group of mice 14 days after treatment, and the proportion of leukemia cells in the peripheral blood was detected by flow cytometry. Three mice from each group were randomly euthanized, and their spleen and bone marrow were collected. The proportion of leukemia cells was detected by flow cytometry, and the spleen was weighed and photographed. The remaining mice were continuously administered the drugs by gavage daily until the control group mice began to die. The administration was then stopped, and the survival and mortality of the mice were observed and recorded, and survival analysis was performed. Figure 4 (A in the middle).

[0058] II. Experimental Results

[0059] Experimental results are as follows Figure 4 As shown, Figure 4Figure B in the diagram shows that, compared to the control group and those treated with avatinib, dasatinib, or imatinib, PZ1 treatment significantly reduced the proportion of peripheral blood leukemia cells in mice. Three mice from each group were randomly euthanized, and bone marrow and spleen cells were collected for flow cytometry analysis. Figure 4 The CE diagram showed that PZ1 significantly reduced the infiltration of leukemia cells in the bone marrow and spleen, while avatinib, dasatinib, or imatinib did not significantly improve the infiltration of leukemia cells in organs. Figure 4 Figure F shows the photographic results of spleen weight and size in each group of mice. The spleen weight of the PZ1 group was significantly lower than that of the control group and other treatment groups. This result indicates that PZ1 inhibits the proliferation of KIT D816V mutant leukemia and can overcome resistance to avatinib, dasatinib, or imatinib.

[0060] In summary, the in vitro studies of this invention show that PZ1 can significantly inhibit the proliferation of various KIT mutation-positive leukemia cells and promote apoptosis at the cellular level, with significantly better effects than existing TKI drugs. Further studies show that PZ1 can stably bind to the KIT protein and inhibit the phosphorylation levels of KIT and its downstream signaling pathways such as STAT5, AKT, and ERK. In vivo studies show that PZ1 can reduce the proportion of leukemia cells in mouse peripheral blood, significantly reduce the infiltration of leukemia cells in the bone marrow and spleen, and reduce spleen weight. Therefore, this invention reveals the therapeutic effect of PZ1 in KIT mutation-positive leukemia and can effectively overcome existing TKI resistance in KIT mutation-positive leukemia, solving the current treatment challenges of drug resistance in KIT mutation-positive leukemia and providing a new strategy for the treatment of this disease.

Claims

1. The application of the small molecule inhibitor PZ1 in the preparation of drugs for treating KIT mutation-positive acute myeloid leukemia, characterized in that, The structural formula of the small molecule inhibitor PZ1 is shown in formula (Ⅰ): ； The KIT mutation is one or more of the following: D816V, N822K, D816F, D816H, D816Y, V560D, and V560G.

2. The application according to claim 1, characterized in that, The KIT mutation-positive acute myeloid leukemia mentioned is drug-resistant KIT mutation-positive acute myeloid leukemia.

3. The application according to claim 2, characterized in that, The drug resistance refers to resistance to avatinib, dasatinib, or imatinib.

4. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by inhibiting the proliferation of leukemia cells and promoting their apoptosis.

5. The application according to claim 1, characterized in that, The drug achieves its therapeutic effect by inhibiting the infiltration of leukemia cells in the bone marrow and spleen.

6. The application according to claim 1, characterized in that, The drug achieves therapeutic effects by inhibiting the phosphorylation levels of KIT and its downstream STAT5, AKT, or ERK signaling pathways.

7. The application according to claim 1, characterized in that, The concentration of the small molecule inhibitor PZ1 in the drug is 1–100 nM.

8. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 1, characterized in that, The drug is available in oral or injectable form.

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