Biomarker for predicting chemosensitivity of acute myelogenous leukemia and application of biomarker

Abstract

The invention provides a biomarker for predicting chemosensitivity of acute myelogenous leukemia and application of the biomarker, and belongs to the technical field of biological medicine. The leptin and fumaric acid provided by the invention can be used as biomarkers for predicting AML chemotherapy resistance. Wherein the leptin influences the chemotherapy sensitivity by regulating metabolic pathways such as mitochondrial OXPHOS and mitochondrial active oxygen mtROS; meanwhile, the invention also discloses a new mechanism that fumaric acid promotes BMSCs to transfer mitochondria to AML cells so as to enhance the OXPHOS level and induce Ara-C drug resistance. The levels of the two are obviously related to the chemotherapy reaction and prognosis stratification of the patient, so that early recognition of the patient with chemotherapy resistance or poor prognosis is facilitated. Leptin and fumaric acid can be detected in bone marrow plasma, the feasibility is high, and clinical transformation is facilitated. By detecting the level of leptin or fumaric acid in bone marrow of a patient, individualized treatment can be achieved, and the survival rate of the patient is increased.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker for predicting chemotherapy sensitivity in acute myeloid leukemia and its application. Background Technology

[0002] Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy, with the highest incidence rate among adult leukemias. Cytarabine (Ara-C) is a key drug in first-line treatment for AML, and drug resistance is a major factor limiting clinical efficacy and leading to disease relapse in AML patients. The bone marrow microenvironment plays a crucial role in the treatment response to AML.

[0003] Therefore, exploring the key mechanisms that determine Ara-C resistance in AML patients from the perspective of the bone marrow microenvironment and establishing highly efficient and specific predictive biomarkers to assess the efficacy of chemotherapy in patients are of great significance for optimizing AML treatment strategies to achieve precision medicine. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide a biomarker for predicting chemotherapy sensitivity in acute myeloid leukemia and its application.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows:

[0006] In a first aspect, the present invention provides the use of a biomarker for predicting chemotherapy sensitivity in acute myeloid leukemia in the preparation of products that predict chemotherapy sensitivity or prognosis in acute myeloid leukemia; said biomarker is leptin or fumaric acid; said chemotherapy sensitivity is the sensitivity of acute myeloid leukemia patients to the chemotherapy drug cytarabine.

[0007] Previous research in this invention found a negative correlation between leptin levels in bone marrow plasma of newly diagnosed AML patients and the clearance rate of bone marrow blasts after the first cycle of chemotherapy. Based on the stratification of initial chemotherapy efficacy, the median leptin level in the non-response (NR) group was significantly higher than that in the objective response rate (ORR) group. Furthermore, patients were divided into high-leptin and low-leptin groups based on median leptin levels. According to the European Leukemia Network 2022 (ELN 2022) risk stratification, the number of patients with good prognosis was significantly higher in the low-leptin group than in the high-leptin group, while the number of patients with poor prognosis was the opposite. These results suggest that leptin levels may be associated with poor prognosis in AML.

[0008] This invention, through the construction of two AML mouse models, MLL-AF9 and AML1-ETO9a, found that leptin does not affect AML progression, but it significantly weakens the killing effect of Ara-C, while the leptin receptor antagonist (Allo-aca) can enhance the effect of Ara-C. In vitro experiments also indicated that leptin does not affect the growth of AML cells, but it can significantly reduce Ara-C chemosensitivity. This suggests that leptin is a key factor in inducing AML chemosensitivity. Furthermore, experiments showed that leptin can significantly upregulate the expression of respiratory chain complex I-related subunits, thereby increasing oxidative phosphorylation (OXPHOS) levels, accompanied by increased production of mitochondrial reactive oxygen species (mtROS). Increased mtROS can stimulate and enhance the antioxidant capacity of AML cells. As an adipokine secreted by adipocytes, leptin can be detected in plasma, enhancing its accessibility as a predictive biomarker.

[0009] In addition, this invention also reveals that fumaric acid is a key metabolic signaling molecule that induces Ara-C chemotherapy resistance in the AML microenvironment.

[0010] Firstly, in two AML mouse models, MLL-AF9 and AML1-ETO9a, the level of fumarate in the bone marrow supernatant was significantly higher than that in healthy controls, and it mainly originated from AML cells themselves, suggesting that AML cells have the ability to actively remodel the bone marrow metabolic microenvironment. Clinical sample analysis further confirmed that the level of fumarate in the bone marrow plasma of newly diagnosed AML patients was significantly negatively correlated with the clearance rate of primitive cells after the first course of Ara-C chemotherapy. The median concentration of fumarate in the NR group was significantly higher than that in the ORR group. According to the ELN 2022 risk stratification, the proportion of patients with poor prognosis in the high fumarate group was significantly higher than that in the low fumarate group, indicating that fumarate level is closely related to poor prognosis in AML. Furthermore, this study consistently demonstrated in in vitro and in vivo experiments using two strategies: drug intervention (exogenous fumarate supplementation) and gene intervention (regulation of SDHA / FH expression in AML cells). Elevated fumarate levels significantly weakened the killing effect of Ara-C, while lowering its levels effectively sensitized chemotherapy.

[0011] Further mechanistic studies revealed that fumaric acid acts on bone marrow mesenchymal stem cells (BMSCs), inducing them to transfer mitochondria to AML cells, ultimately enhancing the OXPHOS level in AML cells and leading to Ara-C chemotherapy resistance.

[0012] Furthermore, compared to peripheral blood, bone marrow fluid is a better representative of the local microenvironment of AML cells. The function and mechanism by which fumaric acid drives Ara-C resistance in bone marrow fluid have been experimentally verified and revealed, thus it can serve as a biomarker for predicting the efficacy and prognosis of chemotherapy in AML patients.

[0013] In a second aspect, the present invention proposes the application of a biomarker for predicting chemotherapy sensitivity in acute myeloid leukemia in the preparation of products for screening chemotherapy-sensitive populations, wherein the biomarker is leptin or fumaric acid; and the chemotherapy-sensitive population is acute myeloid leukemia patients who are sensitive to the chemotherapy drug cytarabine.

[0014] In a third aspect, the present invention proposes the application of substances that detect leptin expression levels or fumarate levels in the preparation of products for detecting chemotherapy resistance to cytarabine in the treatment of acute myeloid leukemia.

[0015] Furthermore, the product for treating chemotherapy resistance in acute myeloid leukemia with cytarabine includes a detection reagent for detecting leptin expression levels or fumarate levels in biological samples.

[0016] Furthermore, the biological sample is bone marrow plasma from a patient with acute myeloid leukemia.

[0017] In a fourth aspect, the present invention proposes the use of substances that inhibit leptin expression and / or substances that inhibit leptin receptor expression and / or leptin receptor antagonists in the preparation of products that enhance the sensitivity of AML cells to cytarabine.

[0018] Furthermore, the leptin receptor antagonist includes Allo-aca.

[0019] In a fifth aspect, the present invention proposes the application of substances that reduce fumarate levels in the preparation of products that enhance the sensitivity of AML cells to cytarabine.

[0020] Compared with the prior art, the present invention has the following significant advantages:

[0021] This invention proposes leptin and fumaric acid as biomarkers for predicting chemotherapy resistance in AML. Leptin influences chemotherapy sensitivity by regulating metabolic pathways such as mitochondrial OXPHOS and mitochondrial reactive oxygen species (mtROS). Simultaneously, it reveals a novel mechanism by which fumaric acid promotes the transfer of mitochondria from BMSCs to AML cells, thereby enhancing OXPHOS levels and inducing Ara-C resistance. Furthermore, the levels of both are significantly correlated with patient chemotherapy response and prognostic stratification, aiding in the early identification of patients with chemotherapy resistance or poor prognosis. Both leptin and fumaric acid can be detected in bone marrow plasma, demonstrating strong feasibility and facilitating clinical translation. By detecting leptin or fumaric acid levels in a patient's bone marrow, personalized treatment can be achieved, improving patient survival rates. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 To analyze the correlation between bone marrow plasma leptin levels and chemotherapy efficacy in newly diagnosed AML patients; Figure A shows a significant negative correlation between bone marrow plasma leptin concentration and bone marrow blast cell clearance rate after the first chemotherapy cycle in newly diagnosed AML patients; Figure B shows the distribution of bone marrow plasma leptin concentration in the ORR and NR groups; Figure C shows the ROC curve of bone marrow plasma leptin used to distinguish between the ORR and NR groups; Figure D shows the bone marrow plasma leptin levels in AML patients stratified according to the ELN 2022 risk classification; Figure E shows the ROC curve of bone marrow plasma leptin distinguishing between favorable and unfavorable risk AML groups; Figure F shows the CCK8 assay results of the activity of blast cells treated with different concentrations of leptin for 24 h or 48 h; Figure G shows the inhibition rate of Ara-C on blast cells after leptin treatment.

[0024] Figure 2 To establish an MLL-AF9 AML mouse model (A), the effects of leptin or leptin receptor antagonist (Allo-aca) on mouse survival (B), liver and spleen size and weight (C), AML cell infiltration (D), and the proportion of AML cells in bone marrow, liver, and spleen (E) were observed.

[0025] Figure 3 To establish the AML1-ETO9aAML mouse model (A), the effects of leptin or leptin receptor antagonist (Allo-aca) on mouse survival (B), liver and spleen size and weight (C), AML cell infiltration (D), and the proportion of AML cells in bone marrow, liver, and spleen (E) were observed.

[0026] Figure 4 Figure 1 shows the results of detecting the total antioxidant capacity of mouse bone marrow AML cells; flow cytometry was used to sort bone marrow AML cells from MLL-AF9AML mice (A) and AML1-ETO9aAML mice (B) and the AML cells were then detected.

[0027] Figure 5 To investigate the effects of leptin on the chemosensitivity and antioxidant capacity of AML cell lines in vitro; Figure A shows the results of CCK8 assay on the effect of leptin on the chemosensitivity of AML cell lines to Ara-C chemotherapy; the results of leptin treatment include total antioxidant capacity (B), GSH / GSSH ratio (C), mRNA levels of antioxidant capacity-related genes (D), catalase activity (E), and superoxide dismutase activity (F).

[0028] Figure 6 Figure 1 shows the proteomic sequencing results of MLL-AF9 mouse bone marrow AML cells; Figure A shows the number of differentially expressed proteins (DEPs) in the mitochondria of MLL-AF9 AML cells; Figure B is a Venn diagram showing overlapping differentially expressed proteins that are upregulated in the leptin + Ara-C group but downregulated in the Allo-aca + Ara-C group; Figure C shows the KEGG pathway enrichment analysis of (B); Figure D is a heatmap analysis of protein expression in the OXPHOS (Ko00190) and ROS (Ko05208) pathways in MLL-AF9 leukemia cells; (E) Gene ontology (GO) term enrichment analysis of upregulated differentially expressed proteins (DEPs) in the leptin + Ara-C group and enrichment analysis of downregulated differentially expressed proteins in the Allo-aca Ara-C group; (F) Heatmap analysis of protein expression in the mitochondrial complex I structural subunits of MLL-AF9 AML cells.

[0029] Figure 7 Figure 1 shows the effects of leptin on mitochondrial respiratory chain complex I activity, OXPHOS levels, and mtROS production in AML cells. Figure 2 shows the results of mitochondrial respiratory chain complex I activity detection after treatment of AML cells with leptin, Allo-aca, or a combination of both. Figure 3 shows the mitochondrial oxygen consumption rate (OCR) and the calculation of basal, maximum, reserve, and ATP in leptin-treated AML cells. Figure 4 shows the representative flow cytometry histograms of mtROS levels and statistical analysis of mean fluorescence intensity in leptin-treated AML cells.

[0030] Figure 8 The results of total antioxidant capacity (A) and chemosensitivity assay in AML cells after treatment with the mtROS-targeting antioxidant Mito-TEMPO alone and in combination with leptin (B).

[0031] Figure 9 Figure 1 shows the effect of LEPR knockout on AML cells; Figure 2 shows the effect of LEPR knockout on AML cells detected by Western blot; Figure 3 shows the representative flow cytometry histogram of mtROS level and statistical analysis of mean fluorescence intensity in LEPR knockout AML cells; Figure 4 shows the detection results of total antioxidant capacity in LEPR knockout AML cells; Figure 5 shows the effect of LEPR knockout on the sensitivity of AML cells to Ara-C chemotherapy by the presence or absence of leptin.

[0032] Figure 10Differences in bone marrow supernatant metabolites between MLL-AF9 and AML1-ETO9aAML mice and healthy mice were identified. Metabolomics analysis was performed on the bone marrow supernatant of healthy mice, MLL-AF9 AML mice, and AML1-ETO9a AML mice: heatmap of relative metabolite content (A), dynamic distribution map of differentially expressed metabolites based on Log2 (Fold Change), showing the 10 differentially expressed metabolites with the smallest P-value (B), and Venn diagram of differentially expressed metabolites (C). High performance liquid chromatography-tandem mass spectrometry was used to detect the fumarate levels in the supernatant of mouse bone marrow stromal cells (mBMSCs) and AML cells isolated from MLL-AF9 AML mice and AML1-ETO9a AML mice (D).

[0033] Figure 11 To analyze the correlation between bone marrow plasma fumarate levels and chemotherapy efficacy in newly diagnosed AML patients; Figure A shows the correlation analysis between bone marrow plasma fumarate concentration and bone marrow blast clearance rate after the first chemotherapy cycle in newly diagnosed AML patients; Figure B shows the distribution of bone marrow plasma fumarate concentration in the ORR and NR groups; Figure C shows the ROC curve of bone marrow plasma fumarate used to distinguish between the ORR and NR groups; Figure D shows the bone marrow plasma fumarate levels in AML patients stratified according to the ELN 2022 risk classification; Figure E shows the ROC curve of bone marrow plasma fumarate distinguishing between favorable and unfavorable risk AML groups.

[0034] Figure 12 An AML mouse model was constructed using the U937 human AML cell line (A). The effects of fumarate (FA) injection on the fumarate level in the bone marrow supernatant of mice (B), the size and weight of the liver and spleen of mice after Ara-C treatment (C), the proportion of AML cells in the bone marrow, liver and spleen (D), and the effect on survival (E) were observed.

[0035] Figure 13 AML mouse models constructed using U937-shCtrl, U937-shSDHA, and U937-shFH (A) were observed. The effects of gene intervention on the fumarate-producing enzyme (SDHA) and catabolic enzyme (FH) of U937 cells on the fumarate level in the bone marrow supernatant of mice (B), the size and weight of the liver and spleen of mice after Ara-C treatment (C), the proportion of AML cells in the bone marrow, liver, and spleen (D), and the survival time (E) were observed.

[0036] Figure 14 To investigate the effect of exogenous fumaric acid on mitochondrial translocation from bone marrow stromal cells to AML cells, HS-5 cells were pretreated with fumaric acid (1000 μM, 48 h), and mitochondria were labeled with MitoTracker Green. These cells were then co-cultured with AML cells for 24 h, and flow cytometry was used to detect the translocation of HS-5 cells to CD45+ cells.+ Mitochondrial translocation levels in AML cells were analyzed using representative flow cytometry histograms (A) and statistical analysis of mean fluorescence intensity (B). AML cells were co-cultured with HS-5 cells pretreated with fumarate (1000 μM, 48 h) for 24 h, and AML cells were isolated. OXPHOS levels were detected using a hippocampal energy metabolism analyzer. Dynamic oxygen consumption rate (OCR) graphs (C) and statistical analysis of basal respiratory volume, maximum respiratory volume, ATP production, and reserve respiratory volume (D) were also performed. Ara-C chemotherapy sensitivity was detected using the CCK8 assay (E). HS-5 is a human bone marrow stromal cell line; U937, THP1, and HL-60 are human AML cell lines.

[0037] Figure 15 To investigate the effect of gene intervention on fumarate-metabolizing enzymes in AML cells on the translocation of bone marrow stromal cells to AML cells mitochondria; HS-5 cells, after mitochondrial labeling with MitoTracker Green, were co-cultured with AML-shCtrl, AML-shSDHA, and AML-shFH cells for 24 h, respectively. Flow cytometry was used to detect the translocation of HS-5 cells to CD45+ cells. + Mitochondrial translocation levels and statistical analysis of mean fluorescence intensity in AML cells (A); AML-shCtrl, AML-shSDHA, and AML-shFH cells were co-cultured with HS-5 cells for 24 h, and AML cells were isolated. Oxygen consumption rate (OCR) was measured using a hippocampal energy metabolism analyzer. Statistical analysis of basal respiratory volume, maximum respiratory volume, ATP production, and reserve respiratory volume was performed (C); Ara-C chemotherapy sensitivity was detected using the CCK8 assay (D). HS-5 is a human bone marrow stromal cell line; U937, THP1, and HL-60 are human AML cell lines; SDHA is succinate dehydrogenase, a fumarate-generating metabolic enzyme; FH is fumarate hydratase. Detailed Implementation

[0038] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0039] Example 1: High levels of leptin in bone marrow plasma of newly diagnosed AML patients are associated with poor response to Ara-C treatment.

[0040] 1. Bone marrow plasma was collected from 84 newly diagnosed AML patients. Plasma leptin levels were measured by ELISA, and patient medical records were also collected. Chemotherapy outcomes were assessed using two main endpoints: bone marrow blast clearance and chemotherapy efficacy. Efficacy was categorized as ORR and NR. Bone marrow clearance was calculated as the percentage reduction in the proportion of blasts from baseline diagnosis (P1) to 4 weeks post-treatment (P2), using the formula: Clearance (%) = [(P1-P2) / P1] × 100%. Chemotherapy efficacy during the hematopoietic recovery period (approximately 4 weeks post-induction therapy) was assessed based on the following criteria: (i) percentage of bone marrow blasts; (ii) hematological recovery status (absolute neutrophil count [ANC] and platelet count); and (iii) extramedullary disease status. ORR included:

[0041] Complete remission (CR): <5% blast cells, no extramedullary lesions, platelets ≥100×10⁻⁶ - / L, ANC≥1.0×10 9 / L);

[0042] Complete remission with incomplete blood cell recovery (CRi: excluding platelets <100×10⁻⁶) 9 / L or ANC < 1.0 × 10 9 (All components outside L meet CR standards);

[0043] Partial remission (PR: 5-25% reduction in blast cells and ≥50% reduction from baseline, platelet / ANC must meet CR criteria);

[0044] NR indicates that the above mitigation criteria have not been met.

[0045] The correlation between leptin levels and primitive cell clearance rate was assessed using Spearman correlation analysis (r-value).

[0046] The results are as follows Figure 1 As shown in Figure A, leptin levels in bone marrow plasma of AML patients were negatively correlated with the clearance rate of bone marrow blasts after the first cycle of chemotherapy. Furthermore, comparative analysis of leptin levels between the ORR and NR groups revealed that the median leptin level in the NR group was significantly higher than that in the ORR group. Figure 1 B). Leptin can accurately predict the efficacy of Ara-C (AUC = 0.84); Figure 1 C). Leptin had the highest concentration in AML with adverse ELN risk and could distinguish between adverse and favorable prognostic diseases with an AUC of 0.81. Figure 1 DE).

[0047] 2. Bone marrow samples from 5 patients were randomly selected. Mononuclear cells were obtained from the bone marrow using Ficoll density gradient centrifugation, and AML cells were sorted by CD45 and CD34 antibody-labeled flow cytometry. The sensitivity of the original AML cells to Ara-C after leptin treatment was detected in vitro. The results showed that leptin itself did not change cell growth in primary leukemia cells. Figure 1 F), but it attenuated the cytotoxicity of Ara-C (F). Figure 1 G).

[0048] The above results indicate that high levels of leptin in newly diagnosed AML patients are associated with poor response to Ara-C chemotherapy and are a poor prognostic factor for AML patients.

[0049] Example 2: Leptin weakens the chemotherapy effect of Ara-C in AML mice

[0050] To investigate the role of leptin in AML chemotherapy, an AML model was established using 8-10 week old C57BL / 6 mice. MLL-AF9 YFP... + Cells are arranged at 2×10 5 Five mice per group were injected intraperitoneally via tail vein. From day 11 to day 20, mice received either leptin (300 ng / kg) or Allo-aca (1 mg / kg) intraperitoneally daily. Subsequently, from day 16 to day 20, mice were concurrently administered Ara-C (100 mg / kg) intraperitoneally. Simultaneously, AML1-ETO9aAML mice were constructed, receiving either leptin or Allo-aca (at the same dosage) from day 14 to day 23, and Ara-C was administered concurrently from day 19 to day 23 to evaluate the efficacy of the combination therapy. Mouse survival, liver and spleen size and weight, and the proportion of AML cells in bone marrow, liver, and spleen were observed.

[0051] In the MLL-AF9 AML mouse model, under treatment with leptin or Allo-aca combined with Ara-C, the following parameters were observed: survival time, liver and spleen size and weight, and the proportion of AML cells in bone marrow, liver, and spleen. Figure 2 As shown; AML1-ETO9aAML mice treated with leptin or Allo-aca combined with Ara-C exhibited varying survival rates, liver and spleen size and weight, and the proportion of AML cells in bone marrow, liver, and spleen. Figure 3 As shown.

[0052] The results showed that in the C57BL / 6 mouse model, leptin could attenuate the killing effect of Ara-C on AML cells, while leptin receptor antagonists could enhance the effect of Ara-C.

[0053] Example 3: Leptin enhances the antioxidant capacity of AML cells

[0054] 1. MLL-AF9 and AML1-ETO9aAML mouse models were constructed, with grouping and treatment methods the same as in Example 2. Flow cytometry was used to sort YFP from the bone marrow of the MLL-AF9 mouse model. + AML cells were used to investigate the effect of leptin on the total antioxidant capacity of AML cells. Results are as follows: Figure 4 The results showed that, compared with the Ara-C treatment alone, the combined effect of leptin enhanced the total antioxidant capacity of AML cells.

[0055] 2. To investigate the effects of leptin on the chemosensitivity and antioxidant capacity of AML cell lines in vitro, three human AML cell lines—U937, HL-60, and THP-1—were used. Leptin (200 ng / mL) was pretreated for 24 h, followed by treatment with 2 × 10⁻⁶ leptin cells. 5 / wells were seeded in a 96-well plate, treated with Ara-C for 24 hours, and then subjected to CCK-8 assay. The results are as follows: Figure 5 The results showed that leptin reduced the inhibition rate of AML cells. Furthermore, after treatment with leptin (200 ng / mL) for 24 h, antioxidant system-related indicators were measured, including: total antioxidant capacity, reduced glutathione / oxidized glutathione (GSH / GSSH), mRNA expression of antioxidant-related genes, and catalase (CAT) and superoxide dismutase (SOD) activities. The results showed that leptin significantly enhanced the antioxidant capacity of AML cells.

[0056] The above results indicate that leptin can enhance the antioxidant capacity of AML cells both in vivo and in vitro.

[0057] Example 4: Leptin activates mitochondrial respiratory chain complex I and increases OXPHOS levels and mtROS production in AML cells.

[0058] 1. Construct an MLL-AF9 AML mouse model. The grouping and treatment methods are the same as in Example 2. Sort bone marrow YFP from the MLL-AF9 mouse model. + AML cells were subjected to proteomic sequencing analysis. Compared with the Ara-C monotherapy group, the leptin combined with Ara-C group showed greater upregulation of mitochondrial proteins, while the Allo-aca combined with Ara-C group showed less upregulation. Figure 6 A); Venn diagrams showed 66 differentially expressed proteins that were upregulated in the leptin + Ara-C group but downregulated in the Allo-aca + Ara-C group. Figure 6 B); Figure 6C shows the KEGG pathway enrichment analysis of (B), and the results show that the differentially expressed proteins are mainly enriched in the OXPHOS and ROS pathways; the heatmap of OXPHOS (Ko00190) and ROS (Ko05208) pathway protein expression in MLL-AF9 leukemia cells shows that most proteins are highly expressed in the leptin combined with Ara-C group. Figure 6 D); Gene ontology (GO) enrichment analysis of differentially expressed proteins (DEPs) upregulated in the leptin + Ara-C group and downregulated in the Allo-acaAra-C group showed that the differentially expressed proteins were all enriched in the assembly of mitochondrial complex I. Figure 6 E); Heatmap analysis of mitochondrial complex I structural subunit protein expression in MLL-AF9 AML cells showed that the complex I structural subunit protein was significantly upregulated in the leptin combined with Ara-C group. Figure 6 F). The above results suggest that leptin significantly activates the OXPHOS and ROS pathways and upregulates the expression of mitochondrial respiratory chain complex I-related subunit proteins.

[0059] 2. In vitro, the activity of complex I and the level of OXPHOS in AML cells treated with leptin (200 ng / mL) for 24 h were detected using a hippocampal energy metabolism analyzer, and the content of mtROS in AML cells was detected using the MitoSox probe. For example... Figure 7 As shown, the activity of complex I was significantly enhanced in AML cells after treatment with leptin, and the levels of OXPHOS and mtROS in AML cells increased.

[0060] Example 5: Leptin enhances the antioxidant capacity of AML cells by increasing mtROS production, thereby inducing AML cell chemotherapy resistance.

[0061] To assess the relationship between leptin-enhanced OXPHOS levels and increased mtROS production and enhanced antioxidant capacity, a mitochondrial superoxide dismutase mimic, Mito-TEMPO, was used to treat AML cells. The total antioxidant capacity and its effect on leptin-induced chemotherapy resistance were assessed. Results are as follows: Figure 8 As shown in the figure, Mito-TEMPO can reverse the increase in total antioxidant capacity of leptin and the decrease in chemotherapy sensitivity.

[0062] Example 6: Knocking out the leptin receptor (LEPR) reduces mtROS production and enhances the effect of Ara-C.

[0063] To evaluate the role of the leptin / LEPR signaling pathway in leptin-mediated chemoresistance in AML, LEPR was knocked out in AML cell lines using the CRPSR / Cas9 system.

[0064] 1. Design three pairs of small guide RNAs (sgRNAs). Two single-stranded oligo DNAs were annealed to form dsDNA, which was then ligated into a GV708 vector that had been pre-digested with restriction endonucleases. The ligation product was transformed into E. coil DH5α competent cells, and single colonies were picked. Transformants were verified by PCR, and positive clones were sent for sequencing. Correct sequences indicated that the LEPR gene knockout vector was successfully constructed.

[0065] 2. Positive recombinant lentiviral plasmids were extracted using the QIAGEN endovirus-free plasmid extraction kit and co-transfected with lentiviral packaging plasmids (psPAX2 and pMD2.G plasmids) into 293T cells for virus packaging. Cell supernatant rich in lentiviral particles was collected, concentrated, and titer tested; a titer ≥1×10⁻⁶ was obtained. 8 Lentiviral solution at TU / mL. The obtained virus was used to infect AML cells. After selection with puromycin for 3–7 days, monoclonal cells were obtained through limiting dilution, and the knockout efficiency was verified by Western blot analysis. Figure 9 A).

[0066] 3. The levels of mtROS, total antioxidant capacity, and chemosensitivity to Ara-C in AML cells with LEPR knockout were examined. This study utilized LEPR knockout in AML cells to block leptin signaling. Results showed that LEPR knockout significantly reduced mtROS levels and total antioxidant capacity, while increasing the chemosensitivity of AML cells to Ara-C. Figure 9 BD).

[0067] Example 7: The level of fumarate in the bone marrow supernatant of AML mice was higher than that in healthy mice, and it mainly originated from AML cells.

[0068] Two AML mouse models, MLL-AF9 and AML1-ETO9a, were established. After the onset of the disease, bone marrow supernatant was collected from the mice, and metabolomic changes in the bone marrow supernatant of AML mice compared with healthy controls were detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS). Changes in all metabolites were visualized using heatmaps. Figure 10 A). Further statistical analysis (P<0.05) was used to screen for differentially expressed metabolites. The results showed that, compared with healthy mice, 5 differentially expressed metabolites were identified in MLL-AF9 AML mice, and 6 differentially expressed metabolites were identified in AML1-ETO9aAML mice. Figure 10 B); These differentially metabolites are mainly enriched in the tricarboxylic acid cycle pathway, with fumarate, citrate, malic acid, and trans-aconitate being consistently upregulated in the bone marrow supernatant of both AML mouse strains. Figure 10C). Bone marrow stromal cells (BMSCs) are an important cellular component of the bone marrow microenvironment in AML. To further investigate the main source of fumarate, AML cells and bone marrow stromal cells from AML mice were further isolated, and their fumarate secretion levels were detected. The results showed that the fumarate secretion level in the supernatant of AML cell culture was higher than that in the supernatant of bone marrow stromal cell culture. Figure 10 D). These results indicate that there is an abnormal accumulation of fumarate in the bone marrow microenvironment of AML mice, and that AML cells are the main source of fumarate.

[0069] Example 8: High levels of fumarate in bone marrow plasma of newly diagnosed AML patients are associated with poor response to Ara-C treatment.

[0070] Bone marrow plasma was collected from 53 newly diagnosed AML patients. High-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect fumarate levels in the plasma, and patient medical records were also collected. Chemotherapy outcome assessment criteria were the same as described in the leptin section.

[0071] The correlation between fumarate levels and primitive cell clearance rate was assessed using Spearman correlation analysis (r-value). Results are as follows: Figure 11 As shown, the level of fumaric acid in bone marrow plasma of AML patients was significantly negatively correlated with the clearance rate of primitive cells after the first course of chemotherapy. Figure 11 A), and by comparing the differences in fumarate levels between the ORR group and the NR group, the results showed that the median fumarate level in the NR group was significantly higher than that in the ORR group ( Figure 11 B); and fumaric acid can accurately predict the efficacy of Ara-C (AUC = 0.8531); Figure 11 C). Fumaric acid had the highest concentration in AML with adverse ELN risk and could distinguish between adverse and favorable prognostic diseases with an AUC of 0.06535. Figure 11 DE).

[0072] The above results indicate that high levels of fumarate in newly diagnosed AML patients are associated with poor response to Ara-C chemotherapy.

[0073] Example 9: Demonstrating through drug and gene intervention that high levels of fumarate weaken the chemosensitivity of AML mice to Ara-C.

[0074] 1. Drug intervention confirms that fumaric acid weakens the efficacy of Ara-C in AML mice: An AML mouse model was established using 5-6 week old NSG immunodeficient mice, and 2.5 × 10⁻⁶ fumaric acid was injected via the tail vein. 5Personally derived AML cell line U937. From day 11 to 15 post-transplantation, mice were administered the following preparations daily via tail vein injection: 0.1% DMSO solvent (200 μL) and fumaric acid (25 mg / kg). From day 15 to 19 post-transplantation, mice were treated with Ara-C (20 mg / kg). Mice survival time, liver and spleen size and weight, and human CD45 levels in bone marrow, liver, and spleen were observed. + The proportion of AML cells was determined, and the level of fumaric acid in mouse bone marrow supernatant was detected.

[0075] This study investigated the effects of fumarate levels on Ara-C treatment response in AML mice, including survival time, liver and spleen size and weight, and human CD45 levels in bone marrow, liver, and spleen. + The proportion of AML cells and the level of fumarate in bone marrow supernatant, such as Figure 12 As shown.

[0076] The results showed that fumaric acid attenuated the cytotoxic effect of Ara-C on AML cells in a mouse model. This was manifested in a significant increase in fumaric acid levels in the bone marrow supernatant of mice treated with fumaric acid, accompanied by increased levels of anti-human CD45 in the bone marrow, liver, and spleen. + The proportion of AML cells increased, hepatosplenomegaly became more pronounced, and survival time was shortened.

[0077] 2. Genetic intervention confirms reduced fumarate-enhancing Ara-C: AML cells are the main source of elevated fumarate levels in bone marrow plasma, and their intracellular fumarate levels are positively correlated with bone marrow plasma fumarate levels. U937 cells with stable knockdown of succinate dehydrogenase A (shSDHA, low fumarate) or fumarate hydratase (shFH, high fumarate) and their control cells (shCtrl) were constructed using shRNA technology. These cells were then divided into 2.5 × 10⁻⁶ cells. 5 The mice were injected via tail vein into 5-6 week old NSG mice. From day 16 to 20 post-transplantation, Ara-C (20 mg / kg) was administered via tail vein. Mice survival, liver and spleen size and weight, and levels of anti-human CD45 in bone marrow, liver, and spleen were observed. + The proportion of AML cells was determined, and the level of fumaric acid in mouse bone marrow supernatant was detected.

[0078] This study investigated the effects of fumarate levels on Ara-C treatment response in AML mice via gene intervention, including survival time, liver and spleen size and weight, and the levels of anti-human CD45 in bone marrow, liver, and spleen. + The proportion of AML cells and the level of fumarate in bone marrow supernatant, such as Figure 13 As shown.

[0079] The results showed that in the AML mouse model, increasing fumarate levels at the AML cell source weakened the killing effect of Ara-C on AML cells, while decreasing fumarate levels enhanced the effect of Ara-C. Specifically, compared with the shCtrl control group, the shSDHA group (low fumarate) mice had lower fumarate levels in the bone marrow supernatant, and after Ara-C treatment, the shSDHA group mice showed reduced splenomegaly and increased levels of anti-human CD45 in the bone marrow and spleen. + The proportion of AML cells remaining was reduced, and survival was prolonged. In contrast, the shFH group (high fumarate) mice showed increased splenomegaly and higher levels of anti-human CD45 in the bone marrow and spleen. + The proportion of residual AML cells increased and the survival time was shortened. In addition, compared with the shCtrl control group, there was no significant difference in the degree of liver enlargement and the proportion of AML cell infiltration in mice in the shSDHA and shFH groups after Ara-C treatment, which may be because Ara-C treatment has essentially cleared AML cells in the liver.

[0080] Example 10: Fumaric acid induces high OXPHOS-related chemotherapy resistance by promoting the mitochondrial translocation of BMSCs to AML cells.

[0081] 1. Drug intervention confirms the role of fumaric acid in promoting mitochondrial translocation and increasing OXPHOS: Increased OXPHOS levels promoted by mitochondrial translocation are an important mechanism by which stromal cells in the bone marrow microenvironment mediate resistance to Ara-C chemotherapy in AML cells. In an in vitro co-culture system, human bone marrow stromal cell line HS-5 was pretreated with fumaric acid, and HS-5 cells labeled with MitoTracker Green fluorescent dye were co-cultured with AML cell lines (U937, THP-1, HL-60). CD45 levels were analyzed by flow cytometry. + The mean fluorescence intensity of MitoTracker Green in AML cells was used to quantify mitochondria translocated from HS-5 cells to AML cells. AML cells were isolated from the co-culture system, and OXPHOS levels in AML cells were detected using a hippocampal energy metabolism analyzer, while Ara-C chemosensitivity was assessed using the CCK8 assay.

[0082] After pretreatment of HS-5 cells with fumarate, the level of mitochondrial transfer from HS-5 cells to AML cells and the sensitivity of AML cells to OXPHOS and Ara-C chemotherapy were observed. Figure 14 As shown.

[0083] The results showed that, compared with the control group, pretreatment of HS-5 cells with exogenous fumaric acid and its derivative dimethyl fumarate (DMF) significantly enhanced the mitochondrial translocation levels into various AML cells. Figure 14AB). Analysis of AML cells isolated from this co-culture system revealed a significantly elevated OXPHOS level (measured using a hippocampal energy metabolism analyzer). Figure 14 CD), while the sensitivity to Ara-C chemotherapy was significantly reduced ( Figure 14 E).

[0084] 2. Genetic intervention confirmed that reducing fumarate weakens mitochondrial translocation and OXPHOS: AML-shCtrl, AML-shSDHA (low fumarate), and AML-shFH (high fumarate) cells were co-cultured with HS-5 cells. CD45 levels were analyzed by flow cytometry. + The mean fluorescence intensity of MitoTracker Green in AML cells was used to quantify mitochondria translocated from HS-5 cells to AML cells. AML cells were isolated from the co-culture system, and OXPHOS levels in AML cells were detected using a hippocampal energy metabolism analyzer, while Ara-C chemosensitivity was assessed using the CCK8 assay.

[0085] After altering fumarate levels through gene intervention, the levels of mitochondrial transfer from HS-5 cells to AML cells and the sensitivity of AML cells to OXPHOS and Ara-C chemotherapy were observed. Figure 15 As shown.

[0086] The results showed that, compared with the AML-shCtrl group, the AML cells in the AML-shSDHA co-culture system had a lower level of mitochondrial translocation ( ). Figure 15 A) OXPHOS levels were significantly reduced ( Figure 15 BC), increased sensitivity to Ara-C chemotherapy ( Figure 15 D). Conversely, the AML-shFH group showed the opposite trend ( Figure 15 AD).

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a biomarker for predicting the chemosensitivity of acute myeloid leukemia in the manufacture of a product for predicting the chemosensitivity or prognosis of acute myeloid leukemia, characterized in that, The biomarker is leptin or fumaric acid; the chemotherapy sensitivity refers to the sensitivity of acute myeloid leukemia patients to the chemotherapy drug cytarabine.

2. Use of the biomarker for predicting the sensitivity of acute myeloid leukemia to chemotherapy in the manufacture of a product for screening a population sensitive to chemotherapy, characterized in that, The biomarker is leptin or fumaric acid; the chemotherapy-sensitive population refers to patients with acute myeloid leukemia who are sensitive to the chemotherapy drug cytarabine.

3. Application of substances that detect leptin expression levels or fumarate levels in the preparation of products for detecting chemotherapy resistance to cytarabine in the treatment of acute myeloid leukemia.

4. The application according to claim 3, characterized in that, The product for treating chemotherapy resistance in acute myeloid leukemia with cytarabine includes a reagent for detecting leptin expression levels or fumarate levels in biological samples.

5. The application according to claim 4, characterized in that, The biological sample was bone marrow plasma from a patient with acute myeloid leukemia.

6. Application of substances that inhibit leptin expression and / or substances that inhibit leptin receptor expression and / or leptin receptor antagonists in the preparation of products that enhance the sensitivity of AML cells to cytarabine.

7. The application according to claim 6, characterized in that, The leptin receptor antagonist includes Allo-aca.

8. Application of substances that reduce fumarate levels in the preparation of products that enhance the sensitivity of AML cells to cytarabine.

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