Application of sorafenib in enhancing anti-leukemia effect of graft

Abstract

The invention discloses application of sorafenib in enhancing the anti-leukemia effect of a graft, and relates to the technical field of biological medicine and clinical medicine.The key point of the technical scheme is that the sorafenib promotes release of IL-18 by activating a Caspase-3 / GSDME dependent pyroptosis pathway of macrophages, so that the GVL effect of NK cells is enhanced. In the actual use process, the expression condition of a macrophage surface marker in peripheral blood or bone marrow of a patient is detected through a flow cytometry method and the like, the patient with high macrophage activity is screened out, and the patient can be more easily benefited from sorafenib treatment, so that precise treatment is realized, and the prognosis of the patient is improved. Layered treatment of patients and drug combination of sorafenib are also realized. The invention can promote the establishment of a corresponding clinical screening, medication and curative effect detection system, provides a new insight for improving maintenance treatment after hematopoietic stem cell transplantation, and has important clinical significance and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine and clinical medicine, and more particularly to application of sorafenib in enhancing graft versus leukemia effect. BACKGROUND

[0002] Acute myeloid leukemia (AML) is a major disease that seriously endangers human life and health, and its clinical manifestations and treatment prognosis have great heterogeneity. FLT3-ITD mutation is the most common gene mutation type of AML, with a high incidence of 25%, and has been confirmed as an independent poor prognostic factor of AML. It is currently believed that allogeneic hematopoietic stem cell transplantation (allo-HSCT) can benefit patients with this type of disease, but the relapse rate after transplantation is still high. The prognosis of patients who relapse after transplantation is extremely poor, and there is a lack of effective treatment methods, which is a current research difficulty.

[0003] Graft versus leukemia (GVL) is the main mechanism of allo-HSCT for curing acute leukemia. Immune cells in allogeneic grafts are the main effector cells that exert GVL, and can also cause graft versus host disease (GVHD). Sorafenib is a multi-target FLT3 inhibitor that can inhibit the activity of different kinases such as FLT3, Raf, c-KIT, vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). Existing technologies show that whether sorafenib is used for induction, consolidation or maintenance treatment before transplantation or used for maintenance or salvage treatment after transplantation can benefit FLT3-ITD positive AML, and this anti-leukemia effect is independent of GVHD, suggesting that sorafenib can induce separation of GVHD and GVL. However, the specific mechanism of sorafenib-induced separation of GVHD and GVL needs to be further elucidated. As a multi-target drug, sorafenib has been shown to activate multiple Caspases, including Caspase-1, 2, 3, 4, 7, 8 and 9, and induce different forms of cell death under different conditions. A study by Hage et al. (Hepatology. 2019; 70(4): 1280-1297) reported that in mice with liver cancer treated with sorafenib, macrophages increased the secretion of IL-18 and IL-1β through Caspase-1-dependent pyroptosis. Although the cleavage of Caspase-1 and the increase in the secretion of cytokines such as IL-1β and IL-18 were also observed in the applicant's previous research, the main effect was achieved through the Caspase-3 / GSDME axis. SUMMARY

[0004] The application aims to provide an application of sorafenib in enhancing a graft versus leukemia effect, discloses that sorafenib promotes the release of IL-18 by activating a Caspase-3 / GSDME-dependent pyroptosis pathway of macrophages, thereby enhancing the GVL effect of NK cells, and on the basis, promotes the establishment of a corresponding clinical screening, medication and efficacy detection system, provides new insights for improving maintenance treatment after hematopoietic stem cell transplantation, and has important clinical significance and application value.

[0005] The above technical purpose of the application is achieved by the following technical scheme: an application of sorafenib in preparing a drug for enhancing a graft versus leukemia effect, the drug being used for patients with high macrophage activity.

[0006] The application is further provided as follows: the sorafenib is used as the only active component of the drug.

[0007] The application is further provided as follows: the drug comprises sorafenib and an exogenous IL-18 or an IL-18 receptor agonist used in combination with sorafenib.

[0008] The application is further provided as follows: the drug does not comprise a broad-spectrum caspase inhibitor.

[0009] The application is further provided as follows: the drug can be used in combination with a glycolysis agonist comprising F1,6BP or F2,6BP.

[0010] In summary, the application has the following beneficial effects:

[0011] In the application, sorafenib promotes the release of IL-18 by activating a Caspase-3 / GSDME-dependent pyroptosis pathway of macrophages, thereby enhancing the GVL effect of NK cells. On the basis, a corresponding clinical screening, medication and efficacy detection system are promoted to be established, new insights are provided for improving maintenance treatment after hematopoietic stem cell transplantation, and important clinical significance and application value are achieved. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 . Cell morphology of M1-like macrophages treated with DMSO or sorafenib for different times; it is observed that the macrophages treated with sorafenib have morphological changes of cell pyroptosis.

[0013] Figure 2 . Scanning electron microscope images of cell morphology of M1-like macrophages treated with different concentrations of sorafenib for 24 h; it is observed that the macrophages treated with sorafenib have morphological changes of cell pyroptosis in different degrees.

[0014] Figure 3. Immunofluorescence analysis of PI staining of Ml-like macrophages after 24 h of treatment with different concentrations of sorafenib; the results show that the death rate of macrophages treated with sorafenib increases in a dose-dependent manner;

[0015] Figure 4 . Analysis of the cytotoxicity of Ml-like macrophages after 24 h of treatment with different concentrations of sorafenib compared to the DMSO group by LDH release assay; the results show that the death rate of macrophages treated with sorafenib increases in a dose-dependent manner;

[0016] Figure 5 . Flow cytometry analysis of the proportion of IFN-γ+ NK cells after 24 h of direct or co-culture with Ml-like macrophages in the presence of sorafenib or DMSO; the results show that inhibition of Caspase-3 but not other Caspases significantly reduces the ability of sorafenib to promote IFN-γ expression in NK cells;

[0017] Figure 6 . Immunoblot analysis of the cleavage of Caspase-3 and GSDME in Ml-like macrophages after 24 h of treatment with different concentrations of sorafenib; the results show that both Caspase-1 and Caspase-3 are cleaved after treatment with sorafenib.

[0018] Figure 7 . Immunoblot analysis of the cleavage of Caspase-3 and GSDME in CD68+ cells from AML patients with or without treatment with sorafenib; cleaved GSDME was detected in a dose-dependent manner and could be reversed by Caspase-3 inhibitors.

[0019] Figure 8 . Cleavage of Caspase-3 and GSDME in Ml-like macrophages after 24 h of treatment with different concentrations of sorafenib with or without Caspase-3 inhibitors; the results show that although cleaved Caspase-3 was detected in patients with or without treatment with sorafenib, cleaved GSDME was only detected in patients treated with sorafenib, regardless of whether they had a FLT3-ITD mutation;

[0020] Figure 9 . IL-18 mRNA levels in Ml-like macrophages and the concentration of IL-18 in the supernatant after 24 h of treatment with 5 μΜ sorafenib or DMSO with or without Caspase-3 inhibitors; the results show that Caspase-3 inhibitors can block the secretion of IL-18 but do not affect its transcription.

[0021] Figure 10. The proportion of K562 cell apoptosis after NK cells were co-cultured with M1-like macrophages treated with Caspase-3 inhibitor for 6h in the presence or absence of sorafenib, AnnexinV-PI staining analysis; the results show that sorafenib cannot promote the cytotoxicity of NK cells in the presence of Caspase-3 inhibitor. DETAILED DESCRIPTION

[0022] The following description will be made in conjunction with the accompanying drawings as follows Figures 1-10 Further detailed description will be made to the present application.

[0023] Sorafenib (C 21 H 16 ClF3N4O3) is a multi-targeted kinase inhibitor, and its CAS number is 284461-73-0.

[0024] The concept of "treatment" in the present application means any measure suitable for treating tumors and related diseases, or prophylactic treatment for such manifested diseases or symptoms, or avoiding recurrence of such diseases, for example, recurrence after the end of the treatment period or treatment of symptoms of already manifested diseases, or pre-emptive intervention to prevent or inhibit or reduce the occurrence of such diseases or symptoms.

[0025] EMBODIMENT

[0026] The test method is as follows:

[0027] (1) Cell culture: the conventional culture of THP1 and K562 cell lines uses RPMI-1640 basic culture medium, and the following components are added to the culture medium: 10% fetal bovine serum, 1% MEM non-essential amino acid solution, 1% 1M HEPES buffer, 1% 100 mM sodium pyruvate, 1% 200 mM L-glutamine, and 1% 100x penicillin-streptomycin mixed anti-mycoplasmic reagent. The cell culture conditions are 37°C constant temperature, stable 5% CO2 incubator.

[0028] (2) Induction of macrophages: For THP1 cells, THP1 were seeded in microplates with medium added with 100 μg / ml PMA and incubated for 24 h to induce M0 macrophages. The cell supernatant was replaced with medium added with 10 ng / ml LPS and 100 ng / ml IFN-γ and incubated for another 24 h to induce M0 macrophages into Ml -like macrophages. For human monocyte-derived macrophages, monocytes were extracted from peripheral blood of healthy donors and cultured in 6-well plates at a density of 1 x 106 cells per well in medium containing 10 ng / ml GM-CSF. Macrophage differentiation required 6 days. The cell supernatant was replaced with medium added with 10 ng / ml LPS and 100 ng / ml IFN-γ and incubated for another 24 h to induce Ml -like macrophages.

[0029] (3) To test the effect of sorafenib on macrophage polarization, we added 5 μΜ sorafenib or DMSO in the culture medium to induce Ml -like macrophages. The cells were tested after 24 h.

[0030] (4) NK cell isolation and expansion in vitro: Mononuclear cells were extracted from peripheral blood of healthy donors or post-transplantation AML patients. NK cells were further isolated using NK cell isolation kit. The purity of NK cells (defined as CD3 CD56+ CD16+) was tested by flow cytometry. NK cells with purity over 85% were expanded for 30 days using OptiVitro® NK cell expansion kit with 10% human serum. Expanded NK cells were generally used 12-21 days after isolation.

[0031] (5) Co-culture of NK cells with macrophages: Isolated or expanded NK cells were washed twice with phosphate buffered saline (PBS) and then co-cultured with induced Ml -like macrophages. For direct co-culture, the Ml macrophage supernatant was discarded and NK cells were resuspended in RPMI-1640 with 10% fetal bovine serum and seeded in microplates containing Ml macrophages at a ratio of 1 : 1. For indirect co-culture, Transwell inserts were used. NK cells and macrophages were loaded into the upper chamber at a ratio of 1 : 1. NK cells and macrophages were co-cultured for 24 ~ 48 h.

[0032] (6) Western blot analysis for Caspase-3 / GSDME pathway activation: THP-1 induced macrophages were washed with PBS twice, lysed with RIPA lysis buffer containing protease inhibitors and protein phosphatase inhibitors on ice for 10 min, centrifuged at 14000 x g, 4°C for 10 min, and the supernatant was used to determine the protein concentration by BCA method and adjusted to be consistent; then 5x loading buffer was added, and the protein was denatured by boiling for 5 min. The samples were subjected to SDS-PAGE gel electrophoresis (such as 7.5%-15% gradient gel) to separate proteins, and then the proteins were transferred to PVDF membranes by wet transfer method. After blocking with 5% skim milk at room temperature for 1 hour, the primary antibody was added and incubated at 4°C overnight; after washing with TBST for 3 times, the secondary antibody was added and incubated at room temperature for 1.5 hours. After washing again, ECL chemiluminescence reagent was used for development, and imaging was performed by electronic imaging system, and finally the band gray value was analyzed by Image J software.

[0033] (7) Isolation of mRNA and quantitative polymerase chain reaction (qPCR) detection of IL-18 mRNA level: The treated macrophages were washed with cold PBS twice, and then total RNA was extracted using TRIzol ™ Reagent. Before qPCR, 1 μg of total mRNA of each sample was reverse transcribed into complementary DNA using HiScript III RTSuperMix for qPCR (+gDNA wiper) reagent kit. Real-time quantitative qPCR was performed using Taq Pro Universal SYBR qPCR Master Mix reagent kit. After M1-like macrophages were treated with 5 μM sorafenib or DMSO for 24 h, the IL-18 mRNA level of M1-like macrophages with or without Caspase-3 inhibitor.

[0034] (8) K562 cytotoxicity test: In order to test whether the pro-inflammatory effect of sorafenib is dependent on pyroptosis induced by Caspase-3 / GSDME, K562 cytotoxicity test was performed. K562 cells were stained with CFSE at 4°C. NK cells were mixed with CFSE-labeled K562 cells at the specified ratio, and cultured for 6 h. Cells were collected and stained using Annexin V-PI kit, and then detected using flow cytometry. At least 10,000 cells were collected for analysis. Total apoptotic cells were CFSE+AnnexinV+, and late apoptotic cells were CFSE+AnnexinV+PI+.

[0035] (9) Enzyme-linked immunosorbent assay (ELISA): After 24 h treatment, macrophage supernatant was collected and centrifuged at 400 x g for 5 min. ELISA kit was used to detect cytokine concentration, and the result was calculated as OD450-OD600. The results of standard samples were subjected to simple linear regression using Graphpad Prism 9.0 software to calculate the concentration. Changes in IL-18 concentration in M1-like macrophage supernatant after 24 h treatment of M1-like macrophages with 5 μΜ sorafenib or DMSO, with or without Caspase-3 inhibitor.

[0036] (10) LDH release assay / PI staining to detect cytotoxicity of sorafenib on macrophages: THP1 was seeded in 96-well plates at a density of 10,000 cells per well and induced into M1-like macrophages. Different concentrations of sorafenib were added to the culture medium, and the cells were cultured for 24 h. LDH release detection used LDH cytotoxicity detection kit. After incubation at room temperature for 30 min, the absorbance was detected at 490 nm. PI staining was performed using Annexin V-PI kit. Images were taken using OLYMPUS IX73 inverted microscope system (OLYMPUS, Tokyo, Japan) and analyzed using ImageJ.

[0037] (11) Light microscopy: THP1 cells were seeded, induced and treated at a density of 20,000 cells per well on 96-well microplates. Photographs and videos were taken at the specified time points after treatment at 400x magnification using the OLYMPUS IX73 inverted microscope system (OLYMPUS, Tokyo, Japan).

[0038] (12) Transmission electron microscopy: THP1 cells were induced into M1-like macrophages and treated, and then collected with a cell scraper, fixed with 2.5% glutaraldehyde at room temperature for 1 hour and then at 4°C overnight. After washing with PBS for 3 times, 1% osmium acid was used for fixation at 4°C for 30-60 min, and then washed with PBS again. After dehydration with ethanol gradient (50%, 70%, 90%, 100%), penetration was performed with epoxy resin and acetone mixture (1:1 volume ratio for 1 hour, 1:2 volume ratio for 2 hours, 1:3 volume ratio for 3 hours) in sequence, and pure resin was used for penetration at room temperature overnight. After embedding of the sample, polymerization was performed at 72°C for 24 hours, and 60-90 nm ultrathin sections were cut with a diamond knife using an ultramicrotome, collected on a 200-mesh copper grid, stained with uranyl acetate and lead citrate, and finally observed and imaged under 5000-10000 times using a JEM-2100 transmission electron microscope.

[0039] (13) Scanning electron microscope: Before seeding THP1, place the evaporated silicon wafer at the bottom of the 24-well microplate hole. After THP1 cells are induced into macrophages and treated with sorafenib / DMSO, the cells attached to the wafer are fixed with 2.5% glutaraldehyde for 10 minutes, and then dehydrated with a series of ethanol solutions with a gradient of 30%-100% volume fraction. The dehydrated sample is dried overnight using a vacuum freeze dryer, and then tested using an AxiaChemiSEM electron microscope under conditions of 5000 to 10000 times.

[0040] In combination with the above experiments and related figures, it is found in the present application that:

[0041] 1. Sorafenib directly inhibits the mitochondrial electron transport chain complex of macrophages, leading to mitochondrial dysfunction (swelling, reduced cristae density, vacuolization), triggering the accumulation of reactive oxygen species (ROS). Morphological changes in macrophage mitochondria are observed by transmission electron microscopy.

[0042] 2. Mitochondrial damage activates Caspase-3, which cleaves pyroptosis key protein GSDME to generate N-terminal fragments (GSDME-N) with membrane perforation ability. GSDME-N induces pyroptosis by forming pores in the cell membrane, resulting in cell swelling, membrane rupture, and LDH release.

[0043] 3. Pyroptosis leads to the release of IL-18 from macrophages, which binds to IL-18R on the surface of NK cells, significantly increasing NK cell IFN-γ secretion and cytotoxicity, enhancing the GVL effect.

[0044] 4. PI staining and lactate dehydrogenase (LDH) release experiments show that cell death rate increases in a dose-dependent manner. Western blotting confirms that sorafenib can activate Caspase-1 and Caspase-3, and that Caspase-3-specific inhibitors significantly inhibit IFN-γ expression in NK cells, suggesting the key role of Caspase-3. Further experiments show that sorafenib induces GSDME cleavage in a dose-dependent manner, and this process can be reversed by Caspase-3 inhibitors, confirming the key role of the Caspase-3 / GSDME axis.

[0045] 5. Clinical sample analysis found that cleaved GSDME was specifically detected in CD68+ macrophages in the bone marrow of AML patients receiving sorafenib maintenance therapy, and Caspase-3 inhibitors could block IL-18 secretion but not affect its transcription. Cytotoxicity experiments showed that the role of sorafenib in enhancing NK cell killing function depends on Caspase-3 activity.

[0046] Since sorafenib promotes the release of IL-18 by activating the Caspase-3 / GSDME-dependent pyroptosis pathway of macrophages, thereby enhancing the GVL effect of NK cells. Therefore, when sorafenib is used for preparing a drug for enhancing the graft versus leukemia effect, the corresponding treatment mode can be selected according to the situation.

[0047] Method one: the expression of macrophage surface markers in the peripheral blood or bone marrow of patients is detected by methods such as flow cytometry, and patients with high macrophage activity are screened out. Such patients may be more likely to benefit from sorafenib treatment, thereby realizing precision treatment and improving the prognosis of patients.

[0048] Method two: by detecting the changes of leukemia cell load, immune function and pyroptosis-related indicators of patients after treatment, the treatment effect and prognosis of patients can be evaluated. For patients with good treatment effect, treatment can be continued; for patients with poor treatment effect or signs of recurrence, the treatment plan is adjusted in time, thereby improving the quality of life and prognosis of patients and realizing the stratified treatment of patients.

[0049] Method three: based on the sorafenib combination regimen found in the present application:

[0050] 1. Combined with exogenous IL-18 or IL-18 receptor agonists, the GVL effect of sorafenib can be enhanced;

[0051] 2. Do not use broad-spectrum caspase inhibitors;

[0052] 3. Combined with glycolysis agonists such as F1,6BP or F2,6BP, this regimen cannot be used for patients who have not achieved CR or have clear recurrence, and other patients can use it.

[0053] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application. Those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, as long as the modifications are within the scope of the claims of the present application and are protected by the patent law.

Claims

1. The application of sorafenib in the preparation of drugs that enhance the anti-leukemia effect of grafts, characterized by: The drug is used for patients with high macrophage activity.

2. The application according to claim 1, characterized in that: Sorafenib is the sole active ingredient in the drug.

3. The application according to claim 1, characterized in that: The drugs include sorafenib and exogenous IL-18 or IL-18 receptor agonists used in combination with sorafenib.

4. The application according to claim 1, characterized in that: The drug does not contain a broad-spectrum caspase inhibitor.

5. The application according to claim 1, characterized in that: The drug can be used in combination with glycolysis agonists, including F1,6BP or F2,6BP.

6. The application according to any one of claims 1-5, characterized in that: The active ingredient in the drug, sorafenib, enhances the GVL effect of NK cells by activating the Caspase-3 / GSDME-dependent pyroptosis pathway in macrophages and promoting the release of IL-18.

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