Kit and method for evaluating relevance between AML NPM1 mutation and Nrf2 based on oxidative stress reaction and application
Through the evaluation method based on oxidative stress response, the association of AML NPM1 mutation and Nrf2 was detected, and the problem of insensitive identification of NPM1 mutant leukemia type and Nrf2 inhibitor efficacy in the prior art was solved, and accurate screening of patients with NPM1 mutant leukemia and the provision of personalized treatment plans were achieved.
Patent Information
- Application Number
- CN202510174647.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to quickly and accurately identify the types of acute myeloid leukemia with NPM1 mutations, and the efficacy of Nrf2 inhibitors is affected by a variety of factors, resulting in insensitivity to treatment in specific patient groups.
The association of AML NPM1 mutations with Nrf2 was detected by an oxidative stress response-based assessment method, including isolating tumor cells, detecting superoxide and Nrf2 protein expression levels, constructing lentiviruses that knock down Nrf2 and NPM1 genes, or using functional inhibitors to screen patients who are sensitive to Nrf2 inhibitors.
Accurate screening of patients with NPM1 mutant leukemia was achieved, subtypes that were truly sensitive to Nrf2 inhibitors were screened out, and personalized treatment plans were provided to provide a basis for the development of new anti-cancer drugs.
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Figure CN119985981A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a kit, method and application for evaluating the correlation between AML NPM1 mutation and Nrf2 based on oxidative stress response. Background Art
[0002] Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease characterized by the accumulation of undifferentiated and uncontrolled proliferation of hematopoietic progenitor cells. Mutations in the nucleophosmin 1 (NPM1) gene are the most common genetic lesions in AML. Leukemia cases with NPM1 mutations exhibit unique biological and clinical features and were therefore recognized as a separate leukemia subtype in the 2017 World Health Organization (WHO) classification of hematopoietic tumors.
[0003] Current treatments for NPM1 mutation leukemia include targeted therapy and immunotherapy. Among them, the molecular targets of targeted therapy include targeting the NPM1 protein structure, the nucleolus of NPM1 mutant cells, the level of NPM1 protein, and targeting other pathways; such as the apoptosis pathway, targeting the MLL-menin complex, and the spleen tyrosine kinase (SYK) signaling pathway. However, most elderly patients with NPM1 mutation leukemia will relapse after treatment and eventually die of progressive diseases. Therefore, NPM1 mutation leukemia urgently needs new strategies to overcome it.
[0004] Existing studies have revealed that tumor cells of acute myeloid leukemia are usually in a state of oxidative stress. However, there are many types of acute myeloid leukemia, and there are no reports on how to quickly and accurately identify the type of NPM1 mutation based on the oxidative stress state of tumor cells.
[0005] Nrf2 is a transcription factor that is mainly responsible for regulating the expression of genes related to cellular antioxidant defense and drug metabolism. Existing studies have revealed that among various leukemia subtypes with high expression of Nrf2, not every subtype of patients is sensitive to treatment with Nrf2 inhibitors (such as ML385). In fact, the efficacy of Nrf2 inhibitors may also be affected by a variety of other factors, including the molecular characteristics of each subtype of leukemia cells themselves, the redundancy of signaling pathways, and the coexistence of other gene mutations. Taking TP53 mutant leukemia as an example, TP53 mutation not only leads to the loss of leukemia cell apoptosis inhibition and DNA repair function, but also may activate other antioxidant and pro-survival signaling pathways to bypass the killing effect of Nrf2 inhibitors. In other words, even in TP53 mutant leukemia with high expression of Nrf2 protein, this subtype of leukemia cells can still maintain their survival and drug resistance through other bypass mechanisms, making them insensitive to treatment with Nrf2 inhibitors.
[0006] In summary, there is an urgent need to quickly and accurately screen specific patient populations suitable for treatment with Nrf2 inhibitors based on the oxidative stress status of tumor cells. Summary of the invention
[0007] In view of this, the purpose of the present invention is to provide a kit, method and application for evaluating the association between leukemia NPM1 mutation and Nrf2 based on oxidative stress response, so as to screen out patients who are sensitive to Nrf2 inhibitors, so as to accurately recommend treatment plans for specific groups of patients. The present invention specifically adopts the following technical solutions.
[0008] The present invention first provides a method for evaluating the association between AML NPM1 mutation and Nrf2 based on oxidative stress response.
[0009] A method for evaluating whether an acute myeloid leukemia patient with high Nrf2 expression has an NPM1 gene mutation based on oxidative stress response, comprising the following steps: S01: Isolate and collect tumor cells from patients with acute myeloid leukemia; S02: (flow cytometry) detecting the superoxide expression level of the tumor cells to obtain a first detection index; the superoxide expression level is a qualitative index; S03: (Western immunoblotting technique) detecting the Nrf2 protein expression level of the tumor cells to obtain a second detection index; the Nrf2 protein expression level includes a qualitative index and a quantitative index; S04: analyzing the first detection index and the second detection index, and when the first detection index is expressed as superoxide expression and the second detection index is expressed as Nrf2 protein expression, determining that the acute myeloid leukemia patient may have high Nrf2 expression and / or NPM1 gene mutation, and continuing verification; S05: constructing a purine-resistant lentivirus that knocks down Nrf2 protein, transfecting the tumor cells with the lentivirus, and then detecting the superoxide expression level of the tumor cells (using flow cytometry) to obtain a third detection index; or, culturing the tumor cells with an Nrf2 protein function inhibitor, and then detecting the superoxide expression level of the tumor cells (using dihydroethidium staining) to obtain a third detection index; The sequence of the small interfering RNA for knocking down Nrf2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8; S06: comparing the first detection index and the third detection index, and when the third detection index is significantly higher than the first detection index, determining that the acute myeloid leukemia patient has high expression of Nrf2 protein; S07: constructing a purine-resistant lentivirus that knocks down the NPM1 gene, transfecting the tumor cells with the lentivirus, and then detecting the Nrf2 protein expression level of the tumor cells (by western blotting) to obtain the fourth detection index; S08: comparing the second detection index and the fourth detection index, and when the fourth detection index is significantly lower than the second detection index, determining that the acute myeloid leukemia patient with high expression of Nrf2 protein has NPM1 gene mutation.
[0010] Further, it is determined that the patient is sensitive to Nrf2 inhibitors.
[0011] Further, the superoxide expression level of the first detection index in S04 is expressed as mean fluorescence intensity, and the mean fluorescence intensity of the first detection index should be no less than 3.7×10 4 .
[0012] Further, the superoxide expression level of the third detection index in S05 is expressed as mean fluorescence intensity, and the mean fluorescence intensity of the third detection index should be no less than 6.4×10 4 .
[0013] In some embodiments, the sequence of the small interfering RNA for knocking down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0014] Another aspect of the present invention provides an application and a kit for evaluating the correlation between AML NPM1 mutation and Nrf2 based on oxidative stress response.
[0015] The invention discloses an application of Nrf2 as a detection marker in the preparation of a kit for evaluating whether an acute myeloid leukemia patient has an NPM1 gene mutation. The kit is used to evaluate whether a leukemia patient with high Nrf2 expression has an NPM1 gene mutation.
[0016] Furthermore, the detection index of the kit includes superoxide expression level.
[0017] Furthermore, in tumor cells of leukemia patients with high Nrf2 expression and NPM1 gene mutation, the superoxide expression level was negatively correlated with Nrf2 gene expression, and the superoxide expression level was positively correlated with NPM1 gene expression.
[0018] Furthermore, the kit contains small interfering RNA and / or an inhibitor of Nrf2 protein function that targets and knocks down the Nrf2 gene; the sequence of the small interfering RNA is shown in SEQ ID NO.7 or SEQ ID NO.8.
[0019] In some preferred embodiments, the Nrf2 protein function inhibitor includes ML385.
[0020] Furthermore, the kit also contains small interfering RNA that targets and knocks down the NPM1 gene; the sequence of the small interfering RNA that targets and knocks down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.2.
[0021] A detection kit for evaluating whether an acute myeloid leukemia patient with high Nrf2 expression has an NPM1 gene mutation based on oxidative stress response, the detection kit comprising a first detection reagent, a second detection reagent and a third detection reagent; The first detection reagent is used to detect the superoxide expression level in the tumor cells of the acute myeloid leukemia patient; The second detection reagent is used to target and knock down the Nrf2 protein in the tumor cells of the acute myeloid leukemia patient; The third detection reagent is used to target and knock down the NPM1 gene in the tumor cells of the acute myeloid leukemia patient; The first detection reagent is dihydroethidium; The second detection reagent is a small interfering RNA that targets and knocks down Nrf2 protein; the sequence of the small interfering RNA that targets and knocks down Nrf2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8; The third detection reagent is a small interfering RNA that targets and knocks down the NPM1 gene; the sequence of the small interfering RNA that targets and knocks down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.2 Beneficial technical effects: 1. The technical solution of the present invention clarifies the correlation between high expression of Nrf2 and NPM1 mutation in leukemia. The present invention first observed the presence of high levels of reactive oxygen species (ROS) in NPM1 mutant leukemia cells, and then verified through experiments that there is a dynamic regulatory relationship between overexpression of antioxidant molecule nuclear factor E2-related factor 2 (Nrf2) and ROS in NPM1 mutant cells. Based on this, the present invention provides multi-dimensional verification, proving that the method of judging whether a leukemia patient has NPM1 mutation based on oxidative stress level is quite innovative, reliable and feasible. Existing studies have revealed that not every leukemia subtype with high expression of Nrf2 is sensitive to Nrf2 inhibitors (such as TP53 mutant leukemia with high expression of Nrf2 protein). The present invention confirms that there is a dynamic regulatory relationship between Nrf2 and ROS in leukemia subtypes that are sensitive to Nrf2 inhibitors. Therefore, the method of the present invention can screen out leukemia subtypes that are truly sensitive to Nrf2, thereby providing a reliable basis for the treatment of patients and an important reference for personalized treatment.
[0022] 2. Based on the correlation between Nrf2 protein and NPM1 mutant protein, the results of the present invention have important implications for the discovery of NPM1 gene mutations. When the test results show that the expression level of Nrf2 protein is abnormally elevated, it can indirectly indicate that the NPM1 gene may be mutated, providing a reference for further gene testing and clinical research. This dual correlation not only enhances the clinical application value of the present method, but also provides new ideas for the molecular mechanism research and targeted treatment of acute myeloid leukemia.
[0023] 3. Based on the correlation between high Nrf2 expression and NPM1 mutation in leukemia verified by the present invention, as well as the dynamic regulatory relationship between Nrf2 and ROS, a more accurate clinical medication regimen is recommended for leukemia patients with NPM1 mutations. For example, Nrf2 inhibitors for inhibiting superoxide expression or the combination of Nrf2 inhibitors and conventional chemical drugs are recommended. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.
[0025] Figure 1 To characterize the relationship between the increased ROS levels and the upregulation of Nrf2 expression in NPM1 mutant cells; Figure 2 To verify the superoxide levels in NPM1-mutated OCI-AML3 cells after knockout of Nrf2 and treatment with the functional inhibitor ML385; Figure 3 To verify the proliferation and apoptosis of OCI-AML3 cells after Nrf2 knockout or ML385 inhibition of Nrf2 function; Figure 4 To verify the apoptosis of OCI-AML3 cells treated with gradient concentrations of ML385 and VEN. DETAILED DESCRIPTION
[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0027] Herein "and / or" includes any and all combinations of one or more of the associated listed items.
[0028] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.
[0029] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.
[0030] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.
[0031] Specific drawings: Figure 1 A shows the superoxide levels in OCI-AML3 cells with NPM1-mA and OCI-AML2 cells with NPM1-wt; it shows that the superoxide levels in NPM1-mutated leukemia cells are higher.
[0032] Figure 1 B is the superoxide level in OCI-AML3 cells with NPM1-mA knockout; it shows that the ROS level in cells was significantly reduced after NPM1 knockout.
[0033] Figure 1 C is the use of qRT-RCR to detect the gene expression of Nrf2 in various leukemia cell lines; it shows that the expression level of Nrf2 in NPM1 mutated leukemia cells is significantly higher than that in other leukemia cells.
[0034] Figure 1 D is the protein expression of Nrf2 in various leukemia cell lines detected by Western Blotting; the results are similar to Figure 1 C matches.
[0035] Figure 1 E: Western Blotting was used to detect the levels of Nrf2 protein in the cytoplasm and nucleus of OCI-AML3 cells after knocking down the NPM1 mutant protein; it shows that the expression of Nrf2 in tumor cells was reduced after knocking down the NPM1 mutant protein.
[0036] Figure 1 F is the relationship between Nrf2 expression and survival outcomes of patients with NPM1 mutation leukemia; it shows that patients with high Nrf2 expression have a lower overall survival rate.
[0037] Figure 2 A: FCM was used to detect the superoxide level in OCI-AML3 cells with NPM1-mA after knocking out the Nrf2 gene; it showed that the ROS level in the cells increased after knocking out the Nrf2 gene.
[0038] Figure 2 B shows the level of superoxide in OCI-AML3 cells treated with 0, 10, and 20 µM ML385 (Nrf2 protein function inhibitor) for 72 hours; the results are similar to Figure 2 A matches.
[0039] Figure 2 C is the expression of Nrf2 protein in OCI-AML3 cells treated with 0, 10, and 20 µM ML385 (Nrf2 protein function inhibitor) for 72 hours; it shows that the expression of Nrf2 protein decreases with increasing concentration.
[0040] Figure 3 A is the apoptosis of OCI-AML3 cells after knocking out the Nrf2 gene using CCK-8 experiment; it shows that the apoptosis rates of cells with different knockdown levels are different, and the greater the knockdown level, the higher the apoptosis rate.
[0041] Figure 3 B shows the apoptosis of OCI-AML3 cells after being treated with different concentrations of ML385 for 72 hours; it shows that as the concentration increases, the cell apoptosis rate increases.
[0042] Figure 3 C is the CCK-8 assay to detect the cell proliferation activity of AML cell lines after treatment with different concentrations of ML385 for 72 hours; it shows that as the concentration increases, the cell proliferation activity decreases.
[0043] Figure 4 A and Figure 4 B shows the apoptosis of OCI-AML3 cells detected by Western Blotting and FCM after 48 hours of combined treatment with gradient concentrations of ML385 and venetoclax (VEN).
[0044] Name explanation: The "AML NPM1 mutation" described in the present invention refers to the type of acute myeloid leukemia with NPM1 mutation.
[0045] The "signal pathway" mentioned in the present invention refers to a series of enzymatic reaction pathways that transmit extracellular molecular signals through the cell membrane into the cell to exert their effects. These extracellular molecular signals (called ligands) include hormones, growth factors, cytokines, neurotransmitters and other small molecule compounds.
[0046] The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The cell culture and statistical analysis methods used in the present invention are as follows.
[0047] Main experimental materials and reagents: (1) Cells: OCI-AML2 and OCI-AML3 cells were purchased from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ).
[0048] Among them, OCI-AML2 cells are NPM1 wild type (marked as NPM1-wt in subsequent experiments); OCI-AML3 cells are NPM1 mutant (marked as NPM1-mA in subsequent experiments).
[0049] (2) Reagents: The culture medium required for the growth of OCI-AML2 cell line is RPMI-1640 culture medium, South American fetal bovine serum and penicillin-streptomycin solution (100 U / mL) at a ratio of 100:10:1. The culture medium required for the growth of OCI-AML3 cells is RPMI-1640 culture medium, Australian fetal bovine serum and penicillin-streptomycin solution (100 U / mL) at a ratio of 100:10:1. The drugs used in the experiment are: proteasome inhibitor MG-132 (Topscience, Shanghai, China, #T2154); Nrf2 inhibitor ML385 (MCE, Monmouth Junction, NJ, USA, #HY-100523); and BCL-2 inhibitor venetoclax (MCE, Monmouth Junction, NJ, USA, #HY-15531).
[0050] (3) Methods: THP-1, MV4-11, OCI-AML2 and OCI-AML3 cells were cultured in an incubator at 37°C and 5% CO2. Cells were passaged every 2-3 days on average to maintain logarithmic growth.
[0051] (4) Statistical analysis: All data were obtained from three independent experiments, and quantitative data were expressed as mean ± standard deviation (± SD). GraphPad Prism (Version 8.0.1) software was used for statistical analysis. Unpaired Student's t-test was used to compare the means of two samples, and one-way analysis of variance was used for pairwise comparisons of multiple groups of data. The test level was α = 0.05, and P < 0.05 indicated that the difference was statistically significant.
[0052] Example 1 This example provides redox state detection in leukemia cells.
[0053] Experimental methods: After treatment with dihydroethidium (DHE) staining probe, flow cytometry was used to analyze the superoxide levels in NPM1-mA OCI-AML3 cells and NPM1-wt OCI-AML2 cells. The detection results were expressed as mean fluorescence intensity (MFI).
[0054] A puromycin-resistant lentivirus was constructed to knock down the NPM1 mutant protein, and then the lentivirus was transfected into OCI-AML3 cells. During the construction process, puromycin was used to screen the cells to construct an OCI-AML3 cell stable strain with knockdown of the NPM1 mutant protein. The above cells were inoculated in 6-well plates with appropriate cell numbers and cultured in an incubator at 37°C and 5% CO2. The cells were collected after 24 hours for subsequent experiments.
[0055] In this example, small interfering RNA was used to knock down NPM1 in OCI-AML3, and the specific information is shown in Table 1.
[0056] Table 1 The experimental results are as follows Figure 1 A and Figure 1 As shown in B, compared with other subtypes of leukemia cells, leukemia cells with high expression of NPM1 mutant protein have higher levels of superoxide (Reactive oxygen species, ROS); after knocking down NPM1 mutant protein, the level of ROS in OCI-AML3 cells decreased. Figure 1 In A, the mean fluorescence intensity of superoxide levels in OCI-AML2 cells was 3.7 × 10 4 The average fluorescence intensity of superoxide level in OCI-AML3 cells was 5.1×10 4 Among them, the vertical axis is the number of cells, and the horizontal axis is the average fluorescence intensity.
[0057] Example 2 Based on Example 1, this example detected the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a key antioxidant regulatory molecule, in NPM1 mutated leukemia cells.
[0058] Experimental method: First, a lentivirus with puromycin resistance that knocks down NPM1 mutant protein was constructed (the method is the same as in Example 1), and then the lentivirus was transfected into OCI-AML3 cells. During the construction process, puromycin was used to screen the cells, and OCI-AML3 stable cell strains with NPM1 mutant protein knockdown were constructed to obtain three OCI-AML3 stable cell strains, shNC, shNPM1#1, and shNPM1#2. The above cells were inoculated in 6-well plates with appropriate cell numbers, and cultured in an incubator at 37°C and 5% CO2. The cells were collected after 24 hours for subsequent experiments.
[0059] Then, real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression level of Nrf2 gene, and Western blotting was used to detect the expression of Nrf2 in THP-1, HL-60, OCI-AML2, OCI-AML3 and OCI-AML3 cells after knocking down NPM1 mutant protein.
[0060] The primer sequences for qPCR detection of Nrf2 involved in this example are shown in Table 2.
[0061] Table 2 The experimental results are shown in Figure 1 C. Figure 1 D and Figure 1 E. The results in the figure show that the Nrf2 gene expression level in OCI-AML3 cells is the highest, which is significantly different from the Nrf2 gene expression levels in the other three leukemia cells; the results of protein immunoblotting experiments show that the Nrf2 protein expression level in OCI-AML3 cells is also the highest. Moreover, after knocking down the NPM1 mutant protein, the Nrf2 protein expression level in OCI-AML3 cells also decreased. This indicates that there is a positive regulatory relationship between NPM1 and Nrf2.
[0062] Example 3 This example, based on Example 2, detected the relationship between Nrf2 expression and the survival outcomes of leukemia patients with NPM1 mutations.
[0063] Experimental methods: Gene expression and clinical characteristics of patients with acute myeloid leukemia were retrieved from the NCBI Gene Expression Omnibus (GEO, https: / / www.ncbi.nlm.nih.gov / gds), access number GSE68466 (n=109), The Cancer Genome Atlas (TCGA, n=86, http: / / www.cancergenome.nih.gov), and Beat AML (http: / / vizome.org / additional_figures_BeatAML.html, n=100) databases. Kaplan-Meier survival analysis was performed using the Kaplan-Meier plotter (http: / / kmplot.com) online tool.
[0064] The experimental results are shown in 1F. The expression of Nrf2 is negatively correlated with the prognosis of NPM1 mutant leukemia patients. That is, the overall survival rate of NPM1 mutant leukemia patients with high Nrf2 expression is lower.
[0065] Example 4 This example detected the regulatory effect of Nrf2 on the redox state of leukemia cells in leukemia with NPM1 mutation.
[0066] Experimental methods: First, a puromycin-resistant lentivirus was constructed to knock down Nrf2 protein, and the lentivirus was transfected into OCI-AML3 cells. During the construction process, puromycin was used to screen the cells to construct a stable strain of OCI-AML3 cells with knockdown of Nrf2 protein. Three stable strains of OCI-AML3 cells, shNC, shNrf2#1, and shNrf2#2, were obtained. After 24 hours of culture, DHE staining and flow cytometry analysis were used to detect the superoxide levels in shNC-OCI-AML3, shNrf2#1-OCI-AML3, shNrf2#2-OCI-AML3, and OCI-AML2 cells; the detection results were expressed as mean fluorescence intensity (MFI).
[0067] In this example, small interfering RNA was used to knock down the Nrf2 gene in OCI-AML3, and the specific information is shown in Table 3.
[0068] Table 3 Furthermore, OCI-AML3 cells were plated at 1.0×10 4 The number of cells was inoculated in a 96-well plate, with 5 replicate wells in each group. The leukemia cells were treated with 0, 10, and 20 μM of the Nrf2 protein function inhibitor ML385 in a concentration gradient manner for 72 h, and the level of intracellular superoxide was measured by DHE staining.
[0069] The experimental results are shown in Figure 2 A and Figure 2 B. As shown in the results, superoxide levels were significantly increased in OCI-AML3 cells with Nrf2 protein knockdown and OCI-AML3 cells treated with ML385, an inhibitor of Nrf2 protein function. Figure 2 In A, the mean fluorescence intensity of superoxide levels in cells knocked down by shNrf2#1 was 6.4×10 4 The mean fluorescence intensity of superoxide levels in cells knocked down by shNrf2#2 was 5.8×10 4 (The vertical axis is the number of cells and the horizontal axis is the average fluorescence intensity). Figure 2 C The results showed that the expression level of Nrf2 protein decreased in OCI-AML3 cells treated with gradient concentrations of ML385, an inhibitor of Nrf2 protein function. The higher the concentration of ML385, the lower the expression level of Nrf2 protein.
[0070] Example 5 This example further detects the effect of inhibiting the Nrf2-mediated antioxidant pathway on the proliferation and apoptosis of OCI-AML3 cells based on Example 4.
[0071] Experimental method: OCI-AML3 cells were cultured at 1.0×10 4 The number of cells was inoculated in a 96-well plate, and 5 replicate wells were set in each group. The apoptosis of shNC-OCI-AML3, shNrf2#1-OCI-AML3, and shNrf2#2-OCI-AML3 cells was analyzed by flow cytometry after being cultured for 72 hours by CCK8.
[0072] Furthermore, OCI-AML3, OCI-AML2, THP-1, and MV4-11 cells were cultured at 1.0 × 10 4 The leukemia cells were treated with Nrf2 protein inhibitor ML385 in a concentration gradient manner for 72 h, and the cell proliferation activity of the leukemia cell lines was detected by CCK8, and the cell apoptosis was analyzed by flow cytometry.
[0073] The experimental results are shown in Figure 3 A and 3B. The results in the figure show that the survival rate of OCI-AML3 cells decreases with the increase of drug concentration under the treatment of ML385. This phenomenon indicates that ML385 can inhibit the Nrf2 protein to promote the increase of ROS levels in leukemia cells, thereby inhibiting tumor cell proliferation and promoting tumor cell apoptosis. The specific experimental results are shown in Tables 4 and 5.
[0074] Table 4 Table 5 The above experimental results show that when shNrf2#2 was used to knock down the Nrf2 gene of OCI-AML3 cells for 72 hours, the cell apoptosis rate was higher than that of shNrf2#1. In addition, its effect was close to that of 10μM ML385.
[0075] Figure 3 C suggests that among the above leukemia cell lines, OCI-AML3 cells are the most sensitive to the Nrf2 protein function inhibitor ML385. This indicates that among leukemia subtypes, leukemia subtypes with NPM1 mutations and high Nrf2 expression are more sensitive to Nrf2 protein function inhibitors.
[0076] However, not every subtype of patients is sensitive to Nrf2 inhibitors (such as ML385), such as TP53 mutant leukemia. Existing studies have revealed that TP53 mutant leukemia with high expression of Nrf2 protein is insensitive to Nrf2 inhibitor treatment (see reference 1).
[0077] Example 6 This example further detects the synergistic anti-tumor effect of ML385 and venetoclax (VEN) on leukemia based on Example 5.
[0078] Experimental method: OCI-AML3 cells were cultured at 1.0×10 4 The number of cells was inoculated in a 96-well plate, and 5 replicate wells were set in each group. In order to detect whether ML385 and the new targeted drug venetoclax (VEN) are superior to the single-drug group in leukemia, OCI-AML3 cells were treated with ML385 and VEN in a concentration gradient for 48 hours, and their cell apoptosis rate was analyzed by flow cytometry and immuno-western blot technology.
[0079] The experimental results are shown in Figure 4 A and Figure 4 B. Combined with the experimental results of Example 5, the leukemia subtype with NPM1 mutation and high expression of Nrf2 is more sensitive to Nrf2 protein function inhibitors; when ML385 and Venetoclax are used in combination, the therapeutic effect of the combination group on leukemia is significantly higher than that of the single-drug treatment group. This shows that for leukemia patients with NPM1 mutations, a low-dose Nrf2 protein function inhibitor combined with a small molecule drug is recommended, which may achieve better tumor remission effects. The results are shown in Table 6.
[0080] Table 6 Example 7 This embodiment provides an example of a method for evaluating whether there is a correlation between Nrf2 gene expression and NPM1 gene expression in leukemia patients based on oxidative stress response.
[0081] S01: From the leukemia patients who come to the hospital for treatment, blood is drawn, and the tumor cells of the patients are separated and collected by conventional experimental means. If necessary, the tumor cells are cultured in the laboratory to reach a quantity that meets the requirements of subsequent testing.
[0082] S02: Detect the superoxide expression level of the tumor cells to obtain a first detection index, which is the superoxide expression level. According to the instruments equipped in the laboratory, the superoxide expression level can be represented by mean fluorescence intensity or other forms commonly used in the art.
[0083] S03: Detecting the Nrf2 protein expression level of the tumor cells to obtain a second detection index, wherein the second detection index is the Nrf2 protein expression amount, which is characterized by a qualitative or quantitative method commonly used in the art.
[0084] S04: Preliminary judgment: when the first detection index is expressed as superoxide expression and the second detection index is expressed as Nrf2 protein expression, it is determined that the leukemia patient may have high Nrf2 expression and / or NPM1 gene mutation, and further verification is performed.
[0085] S05: constructing a purine-resistant lentivirus for knocking down Nrf2 protein (the lentivirus can be constructed in advance by standardized means to meet the needs of real-time detection. The sequence of the small interfering RNA targeting the knockdown of Nrf2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8), and after transfecting the tumor cells with the lentivirus, detecting the superoxide expression level of the tumor cells by flow cytometry to obtain the third detection index; or, after culturing the tumor cells with an Nrf2 protein function inhibitor, detecting the superoxide expression level of the tumor cells by dihydroethidium staining to obtain the third detection index.
[0086] S06: comparing the first detection index and the third detection index, and when the third detection index is significantly higher than the first detection index, determining that the leukemia patient has high expression of Nrf2 protein.
[0087] S07: constructing a purine-resistant lentivirus for knocking down the NPM1 gene (the lentivirus can be constructed in advance using standardized means to meet the needs of real-time detection. The sequence of the small interfering RNA targeting the knockdown of the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.2), and after transfecting the tumor cells with the lentivirus, detecting the Nrf2 protein expression level of the tumor cells by protein immunoblotting technology to obtain the fourth detection indicator.
[0088] S08: Final judgment, comparing the second detection index and the fourth detection index, when the fourth detection index is significantly lower than the second detection index, that is, when the patient's tumor cells show a reduced level of Nrf2 protein expression after targeted knockout of the NPM1 gene, it proves that the patient has a correlation between high Nrf2 expression and NPM1 mutation, and finally determines that the leukemia patient has an NPM1 gene mutation; further, it can be determined that the patient is sensitive to Nrf2 inhibitors.
[0089] Based on the above detection, the correlation between high expression of Nrf2 and NPM1 mutation in a specific patient population was clarified by using oxidative stress response as a detection index. In the aforementioned embodiments, the present invention has confirmed that leukemia NPM1 cells with high expression of Nrf2 are sensitive to Nrf2 inhibitors, and the apoptosis rate of the cells increases after using ML385. Nrf2 is mainly responsible for regulating the antioxidant defense of cells. Therefore, this method can not only screen out people suitable for treatment with Nrf2 inhibitors, providing an important basis for personalized treatment; it can also provide a basis for the development of anticancer drugs or reagents that inhibit superoxide expression.
[0090] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.
[0091] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.
[0092] References: (1)Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative Stress in Cancer. Cancer Cell . 2020;38(2):167-197. doi:10.1016 / j.ccell.2020.06.001IF:48.8 Q1
Claims
1. A method for evaluating whether an acute myeloid leukemia patient has NPM1 gene mutation based on Nrf2 expression based on oxidative stress response, characterized in that: The following steps are involved: S01: Isolate and collect tumor cells from patients with acute myeloid leukemia; S02: The superoxide expression level of the tumor cells is used to obtain a first detection index; the superoxide expression level is a qualitative index; S03: detecting the Nrf2 protein expression level of the tumor cells to obtain a second detection index; the Nrf2 protein expression level includes a qualitative index and a quantitative index; S04: analyzing the first detection index and the second detection index, and when the first detection index is expressed as superoxide expression and the second detection index is expressed as Nrf2 protein expression, determining that the acute myeloid leukemia patient may have high Nrf2 expression and / or NPM1 gene mutation, and continuing verification; S05: constructing a purine-resistant lentivirus that knocks down Nrf2 protein, transfecting the tumor cells with the lentivirus, and detecting the superoxide expression level of the tumor cells to obtain a third detection index; or, culturing the tumor cells with an Nrf2 protein function inhibitor, and detecting the superoxide expression level of the tumor cells to obtain a third detection index; The sequence of the small interfering RNA for knocking down Nrf2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8; S06: comparing the first detection index and the third detection index, and when the third detection index is significantly higher than the first detection index, determining that the acute myeloid leukemia patient has high expression of Nrf2 protein; S07: constructing a purine-resistant lentivirus that knocks down the NPM1 gene, transfecting the tumor cells with the lentivirus, and then detecting the Nrf2 protein expression level of the tumor cells to obtain the fourth detection index; S08: Compare the second detection index and the fourth detection index. When the fourth detection index is significantly lower than the second detection index, determine that the acute myeloid leukemia patient with high expression of Nrf2 protein also has NPM1 gene mutation.
2. The method according to claim 1, characterized in that The superoxide expression level of the first detection indicator in S04 is represented by the mean fluorescence intensity, and the mean fluorescence intensity of the first detection indicator is not less than 3.7×10 4 .
3. The method according to claim 1, characterized in that The superoxide expression level of the third detection indicator in S05 is represented by the mean fluorescence intensity, and the mean fluorescence intensity of the third detection indicator is not less than 6.4×10 4 .
4. The method according to claim 1, wherein: The sequence of the small interfering RNA for knocking down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.
2.
5. Use of Nrf2 as a detection marker in the preparation of a kit for evaluating whether an acute myeloid leukemia patient has an NPM1 gene mutation, characterized in that: The kit is used to evaluate whether a leukemia patient with high Nrf2 expression has an NPM1 gene mutation.
6. The use according to claim 5, characterized in that The detection index of the kit includes superoxide expression level.
7. The use according to claim 6, characterized in that In tumor cells of leukemia patients with high Nrf2 expression and NPM1 gene mutation, superoxide expression level was negatively correlated with Nrf2 gene expression, and superoxide expression level was positively correlated with NPM1 gene expression.
8. The use according to claim 5, characterized in that The kit contains small interfering RNA and / or an inhibitor of Nrf2 protein function that targets and knocks down the Nrf2 gene; the sequence of the small interfering RNA is shown in SEQ ID NO.7 or SEQ ID NO.
8.
9. The use according to claim 5, characterized in that The kit also contains small interfering RNA that targets and knocks down the NPM1 gene; the sequence of the small interfering RNA that targets and knocks down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.
2.
10. A detection kit for evaluating whether an acute myeloid leukemia patient with high Nrf2 expression has NPM1 gene mutation based on oxidative stress response, characterized in that: The detection kit comprises a first detection reagent, a second detection reagent and a third detection reagent; The first detection reagent is used to detect the superoxide expression level in the tumor cells of the acute myeloid leukemia patient; The second detection reagent is used to target and knock down the Nrf2 protein in the tumor cells of the acute myeloid leukemia patient; The third detection reagent is used to target and knock down the NPM1 gene in the tumor cells of the acute myeloid leukemia patient; The first detection reagent is dihydroethidium; The second detection reagent is a small interfering RNA that targets and knocks down Nrf2 protein; the sequence of the small interfering RNA that targets and knocks down Nrf2 protein is shown in SEQ ID NO.7 or SEQ ID NO.8; The third detection reagent is a small interfering RNA that targets and knocks down the NPM1 gene; the sequence of the small interfering RNA that targets and knocks down the NPM1 gene is shown in SEQ ID NO.1 or SEQ ID NO.2.