Use of leucine in preparation of a drug for inhibiting proliferation of hematological tumor cells and inducing apoptosis of hematological tumor cells

Abstract

This invention provides the application of leucine in the preparation of drugs that inhibit the proliferation of hematologic malignancies and induce apoptosis in hematologic malignancies, belonging to the field of biomedical technology. The hematologic malignancies include lymphoid malignancies, specifically B-cell precursor acute lymphoblastic leukemia, B-cell lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. This invention also provides the application of leucine in the preparation of drugs that improve the clinical prognosis of patients with hematologic malignancies and as adjuvant drugs for CAR-T cell therapy of hematologic malignancies. This invention is the first to discover and demonstrate that leucine has a significant antitumor effect and prognostic improvement effect on hematologic malignancies, with a clear mechanism of action, high safety, no obvious toxic side effects, improved patient prognosis, and prolonged survival, thus expanding the pharmaceutical applications of leucine and providing a new direction for the treatment of hematologic malignancies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of leucine in the preparation of drugs that inhibit the proliferation of hematologic tumor cells and induce apoptosis of hematologic tumor cells. Background Technology

[0002] Lymphoid malignancies (such as lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma) are among the most prevalent malignant tumors globally, with their incidence rate increasing year by year, placing a heavy burden on society and healthcare. Current clinical treatments for hematologic malignancies mainly include chemotherapy, targeted therapy, CAR-T cell therapy, and hematopoietic stem cell transplantation. However, existing treatment options have several limitations: First, chemotherapy and targeted therapy are prone to drug resistance and have significant toxic side effects on normal hematopoietic and immune cells, leading to adverse reactions such as bone marrow suppression and weakened immunity. Second, while CAR-T cell therapy is effective for some B-cell lymphoma patients, some still have poor treatment responses and poor prognoses, lacking effective adjuvant therapies to improve treatment outcomes. Third, current clinical interventions primarily focus on directly killing tumor cells; safe drugs have not yet been developed to inhibit or assist in inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and improving overall patient prognosis. Summary of the Invention

[0003] In view of the technical shortcomings of existing treatments for hematologic malignancies, such as high toxicity and side effects, easy drug resistance, and lack of prognostic improvement methods for CAR-T therapy, this invention provides the application of leucine in the preparation of drugs that inhibit the proliferation of hematologic malignancies and induce apoptosis of hematologic malignancies.

[0004] The objective of this invention is achieved through the following technical solution: This invention provides the application of leucine in the preparation of drugs that inhibit the proliferation of hematologic malignancies and induce apoptosis in hematologic malignancies, wherein the hematologic malignancies include lymphoid malignancies, specifically B-cell precursor acute lymphoblastic leukemia, B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, etc.

[0005] This invention also provides the use of leucine in the preparation of medicaments that improve the clinical prognosis of patients with hematologic malignancies, the medicaments being suitable for patients with non-Philadelphia B-cell acute lymphoblastic leukemia and patients with B-cell lymphoma receiving CAR-T cell therapy.

[0006] This invention also provides the application of leucine in the preparation of adjuvant drugs for CAR-T cell therapy of hematologic malignancies.

[0007] In this invention, the drug uses leucine as the core active ingredient, and can be composed of leucine alone or compounded with edible excipients. The dosage forms include powder, granules, capsules, and oral liquid.

[0008] In this invention, the drug contains 90-98 parts by weight of leucine and 5 parts by weight of mannitol.

[0009] This invention is the first to discover and confirm that leucine has a significant antitumor effect and prognostic improvement effect on hematologic malignancies, and compared with existing hematologic malignancy treatment technologies, it has the following significant beneficial effects: 1. Clear mechanism of action and significant antitumor effect: Leucine exerts its antitumor effect by inhibiting the FASN / SREBF1 pathway to reprogram lipid metabolism. Figure 5 It directly inhibits tumor cell proliferation and induces apoptosis, and its clear antitumor effect has been verified in cell lines such as B-cell precursor acute lymphoblastic leukemia and B-cell lymphoma, as well as in animal models.

[0010] 2. High safety and no obvious toxic side effects: Leucine is an essential amino acid for the human body. Intervention through oral supplementation will not produce toxic side effects on the patient's normal hematopoietic and immune cells, avoiding the adverse reactions such as bone marrow suppression and low immunity of traditional methods such as chemotherapy and targeted therapy. It is suitable for long-term intervention for patients.

[0011] 3. Improve patient prognosis and prolong survival: In patients with non-Philadelphia B-cell acute lymphoblastic leukemia, higher plasma leucine levels are associated with better clinical prognosis; in B-cell lymphoma patients receiving CAR-T cell therapy, leucine can significantly prolong overall survival and progression-free survival, filling the gap in adjuvant methods for improving prognosis after CAR-T therapy.

[0012] 4. Expanded the medicinal applications of leucine and provided a new direction for the treatment of hematologic malignancies: Breaking through the traditional understanding of leucine as only a basic nutrient, it has been developed into a therapeutic drug for hematologic malignancies, providing a new and safe adjuvant therapy for clinical use, with broad clinical application prospects. Attached Figure Description

[0013] Figure 1 shows that high-leucine culture inhibits the proliferation of hematologic malignancies and induces apoptosis. Figures A and C are flow cytometry results of Annexin V / 7-AAD staining. Compared with conventional culture (50 mg / L leucine), high-leucine culture (550 mg / L) significantly induced apoptosis in Nalm6 (B-ALL) and SU-DHL-8 (DLBCL) cells. The proportions of each apoptotic subset (Q1–Q4) are marked in the figures. Figures B and D are proliferation curves detected by the Incucyte® S3 live cell analysis system. High-leucine culture significantly inhibited the time-dependent proliferation of both cell types. Cell numbers were normalized relative to baseline (day 0) (n = 3 independent experiments, mean ± standard error).

[0014] Figure 2 shows the effects of a high-leucine diet on the survival time and peripheral blood parameters of model mice. Figure A shows the survival status of each group of BCP-ALL leukemia mouse models, and Figure B shows the changes in hemoglobin (Hb) and platelet (PLT) levels in each group of BCP-ALL leukemia mouse models.

[0015] Figure 3 shows the association analysis between leucine and the prognosis and risk of non-Philadelphia positive B-cell acute lymphoblastic leukemia. Figure (A) shows the prognostic survival curves of the ALL cohort (n=36 per group), indicating that patients with non-Philadelphia positive B-cell acute lymphoblastic leukemia (B-ALL) with higher plasma leucine levels had significantly higher survival rates than those with lower levels (p=0.045), suggesting a positive correlation between leucine levels and patient prognosis. The horizontal axis represents follow-up days, and the vertical axis represents survival rate. (B) The differential expression volcano plot shows the expression changes of leucine and other metabolites. The horizontal axis represents the Log2 fold change (i.e., Log2FC, where fold change refers to the fold difference in leucine levels between lymphocytic leukemia individuals and healthy controls), and the vertical axis represents -Log2FC. 10 (P-value), the figure shows the trend of L-leucine expression changes (orange dots). Gray dots represent significantly downregulated metabolites (FC<1, p<0.05); blue dots represent significantly upregulated metabolites (FC>1, p<0.05); orange dots represent metabolites with no significant change. Figure (C) shows the results of Mendelian randomization analysis using an inverse variance weighted (fixed effects) model, where gene-predicted elevated plasma leucine levels were a protective factor against lymphocytic leukemia (odds ratio OR=0.59, 95% confidence interval 0.35–0.99, P=0.046). The horizontal axis represents the effect size, with the left side indicating the protective factor and the right side indicating the risk factor.

[0016] Figure 4 shows the impact of leucine on the survival of patients receiving CAR-T cell therapy. This figure is a forest plot, illustrating the association analysis results between leucine exposure and the prognosis of B-cell lymphoma patients receiving CAR-T cell therapy. The hazard ratio (HR) and its 95% confidence interval (95% CI) were used as evaluation indicators. The horizontal axis, from left to right, represents the directions of protective factors and risk factors, respectively. The dashed line at 1.0 is the hazard ratio reference line.

[0017] Figure 5 This study demonstrates that leucine exerts its antitumor effect by inhibiting the FASN / SREBF1 pathway reprogramming lipid metabolism. Figure A shows a heatmap of differentially expressed genes (DEGs) in NALM6 cells under standard culture conditions (50 mg / L leucine) and high-leucine culture conditions (550 mg / L leucine). Figures B1-B3 show the results of gene set enrichment analysis (GSEA) of the adipogenic differentiation pathway based on the Reactome database. Compared to the normal control group, adipogenic differentiation-related pathways were significantly inhibited under high-leucine conditions (Reactome database). The horizontal axis represents gene sequence position, the upper vertical axis represents the enrichment fraction change curve, and the lower vertical axis represents gene sequence index. Figure C shows that high-leucine culture significantly downregulated the mRNA expression levels of key lipid metabolism enzymes SREBF1 and FASN in NALM6 cells ("**" indicates P<0.01, "*" indicates P<0.05). Figures D1-D2 show the results of annexin V flow cytometry. C75 treatment (20–40 μM) can induce dose-dependent apoptosis in NALM-6 cells, with a significantly increased proportion of early apoptosis (Q3) and late apoptosis / death (Q2) cells.

[0018] The above figures can all be obtained through specific implementation experiments of the present invention, clearly demonstrating the antitumor effect and prognostic improvement effect of leucine on hematologic malignancies. Detailed Implementation

[0019] Example 1: A Study on the Metabolic, Causal, and Clinical Association of Leucine with Lymphocytic Leukemia 1. Screen for differentially expressed metabolites between patients with lymphocytic leukemia and healthy controls, and identify key differentially expressed molecules. (1) Samples and grouping: Serum samples were selected from 75 patients with acute lymphoblastic leukemia B and 12 healthy controls at the National Center for Translational Medicine (Shanghai).

[0020] (2) Metabolomics detection: Metabolomics technology (LC-MS / MS) was used to perform full metabolite profile analysis on the sample to obtain the quantitative signal intensity of metabolites.

[0021] (3) Data processing: Normalize, logarithmically transform and statistically test the raw data, and calculate the fold change (FC) and p value of each metabolite between the two groups.

[0022] The results are as follows Figure 3 B,L-leucine was significantly downregulated in patients with B-cell acute lymphoblastic leukemia (Log2FC<0, p<0.05), making it a key differential metabolite associated with the disease.

[0023] 2. Causal association analysis between leucine levels and the risk of developing lymphocytic leukemia (1) Experimental grouping and experimental design We used a two-sample Mendelian randomization (MR) approach to assess the potential causal association between circulating leucine levels and the risk of developing lymphoid leukemia.

[0024] Exposure factor data were obtained from the publicly available Genome-wide Association Study (GWAS) database of leucine levels, with a total sample size of 115,078 individuals from the European population. Outcome event data were obtained from the Lymphocytic Leukemia GWAS database, which included 1,493 patients with lymphocytic leukemia and 299,952 controls, for a total sample size of 301,445. All participants were of European descent.

[0025] Using genetic variant sites (SNPs) that are significantly associated with circulating leucine levels as instrumental variables, leucine levels as an exposure factor, and lymphocytic leukemia as an outcome event, a two-sample Mendelian randomization analysis model was constructed to infer the causal relationship between the two.

[0026] (2) Experimental steps ①GWAS Data Acquisition Download the GWAS summary statistics related to leucine levels from the OpenGWAS database.

[0027] Exposure factors: Leucine levels (phenotype number met-c-897), and pooled statistics of lymphocytic leukemia-related GWAS were downloaded from the FinnGen R9 database.

[0028] Ending event: Lymphoid leukaemia, data source address is as follows: https: / / storage.googleapis.com / finngen-public-data-r9 / summary_stats / finngen_R9_CD2_LYMPHOID_LEUKAEMIA_EXALLC.gz.

[0029] ② Instrumental variable selection Single nucleotide polymorphisms (SNPs) that are significantly associated with leucine levels are screened as instrumental variables and linkage disequilibrium (LD) pruning is performed to ensure that each SNP is independent of the others.

[0030] ③ Data Harmonization Allele orientation was unified for GWAS data of exposure factors and outcome events, and inconsistent and palindromic SNPs were removed to ensure consistency of effect orientation.

[0031] ④ Mendelian randomization analysis The main causal effects were estimated using the inverse variance weighted (IVW) fixed effects model, and the odds ratio (OR), 95% confidence interval (95% CI) and p-value were calculated.

[0032] ⑤ Results visualization Forest plots were created to illustrate the estimated MR effect between leucine levels and the risk of lymphocytic leukemia.

[0033] Results (Figure 3C): Elevated circulating leucine levels were significantly associated with a reduced risk of developing lymphocytic leukemia (OR=0.59, 95% CI=0.35~0.99, p=0.046), suggesting that leucine is a protective factor against lymphocytic leukemia.

[0034] 3. Assess the correlation between leucine expression levels and prognosis in patients with lymphocytic leukemia. In the non-Ph B-ALL patient cohort of the National Center for Translational Medicine (Shanghai), a total of 72 non-Ph B-ALL patients were included, all from consecutive enrollments at the same center. Based on baseline plasma leucine levels (the initial concentration of leucine in the blood of the subjects before the start of the study), patients were grouped according to the median: a high leucine expression group (patients with an initial blood leucine concentration ≥ the median, n=36) and a low leucine expression group (patients with an initial blood leucine concentration < the median, n=36). Peripheral blood samples were collected from patients, and leucine concentration was quantitatively detected using liquid chromatography-mass spectrometry (LC-MS) or targeted metabolomics methods. Patients were followed up long-term, and follow-up time and survival outcomes were recorded. Overall survival (OS) was assessed using the Kaplan-Meier method.

[0035] The results are as follows Figure 3A. The survival rate of patients in the high leucine expression group was significantly higher than that in the low expression group (Log-rank test, p=0.045), suggesting a positive correlation between leucine level and patient prognosis. Patients with higher plasma leucine levels (≥median) had a 2-year overall survival rate of over 70%, which was higher than that of patients with lower plasma leucine levels (<median, 2-year overall survival rate <40%).

[0036] 4. Evaluate the impact of leucine on the efficacy (progression-free survival, overall survival) of CAR-T cell therapy in patients with diffuse large B-cell lymphoma (DLBCL). (1) Data sources and experimental groups A secondary analysis was performed based on publicly available CAR-T therapy-related metabolomics data. The original study subjects were patients with relapsed / refractory large B-cell lymphoma (r / r LBCL) who received anti-CD19 CAR-T cell therapy. The relevant plasma metabolomics data were obtained from publicly available literature and its associated datasets (Fahrmann, JF et al. A polyamine-centric, blood-based metabolite panel predictive of poor response to CAR-T cell therapy in large B cell lymphoma. Cell reports. Medicine 3, 100720 (2022). https: / / doi.org / 10.1016 / j.xcrm.2022.100720).

[0037] The original study grouped patients based on their clinical outcomes after CAR-T therapy, including: ① ongoing complete response (CR); ② progressive disease / partial response (PD / PR). Progression-free survival (PFS) and overall survival (OS) were used as the primary clinical endpoints.

[0038] (2) Data analysis process Leucine-related metabolic features were extracted from publicly available metabolomics data, and their association with the prognosis of CAR-T therapy was further analyzed.

[0039] Based on patient survival outcome information, the Cox proportional hazards regression model was used to assess the relationship between circulating leucine levels and progression-free survival (PFS) and overall survival (OS), and the hazard ratio (HR) and 95% confidence interval (95% CI) were calculated.

[0040] Further forest plots were created to demonstrate the correlation between leucine and the efficacy of CAR-T therapy.

[0041] The results are shown in Figure 4. The results showed that plasma leucine levels were significantly associated with improvements in progression-free survival (PFS) and overall survival (OS). Hazard ratio analysis indicated that leucine was a prognostic protective factor (HR<1, confidence interval was generally to the left of the 1.0 reference line), suggesting that leucine can reduce the risk of disease progression and death in patients and improve the efficacy of CAR-T cell therapy.

[0042] Example 2: In vitro tumor-suppressing experiment of leucine on hematologic tumor cells (a) Experimental materials 1. Tumor cell lines: The NALM6 cell line, a B-cell precursor acute lymphoblastic leukemia, and the SU-DHL-8 cell line, a B-cell lymphoma, were both purchased from the ATCC cell bank; 2. Leucine: Leucine powder with a purity of ≥98% (Targetmol); 3. Cell culture medium: RPMI-1640 complete medium, prepared as follows: Add 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin to the RPMI-1640 (Shanghai Yuanpei Biotechnology) basal medium. The penicillin and streptomycin are penicillin and streptomycin respectively. The concentration of penicillin in the RPMI-1640 complete medium is 100 U / mL and the concentration of streptomycin is 100 μg / mL.

[0043] 4. Detection kits: Annexin-V / 7-AAD apoptosis detection kit (Thermo), CCK-8 proliferation detection kit (Thermo).

[0044] 5. Experimental instruments: flow cytometer, Incucyte® S3 live cell imaging analysis system, clean bench, carbon dioxide incubator.

[0045] (II) Experimental Methods 1. Cell Culture NALM6 and SU-DHL-8 cells were placed in carbon dioxide incubators at 37°C and 5% CO2, respectively, and cultured routinely in RPMI-1640 complete medium. Cells in the logarithmic growth phase were used for experiments.

[0046] 2. Leucine treatment NALM6 cells were treated with leucine as follows: Drug-containing culture media were prepared, with a normal control group (RPMI-1640 complete medium containing leucine at a concentration of 50 mg / L, the standard cell culture concentration) and a high-leucine group (RPMI-1640 complete medium supplemented with leucine to achieve a concentration of 550 mg / L). NALM6 tumor cells in logarithmic growth phase were collected, and the cell density was adjusted to obtain 1 × 10⁶ cells / year. 5 100 μL of the cell suspension was added to each well of a 96-well plate (for cell proliferation detection) and 2 mL of the cell suspension was added to each well of a 6-well plate (for apoptosis detection). After cell seeding, the 96-well and 6-well plates were set up as a normal control group and a high-leucine group, respectively, and the corresponding culture medium was added. Each group was divided into 3 replicates and incubated in a 37°C, 5% CO2 incubator.

[0047] SU-DHL-8 cells were treated with leucine using the same method.

[0048] 3. Apoptosis detection After culturing NALM6 and SU-DHL-8 cells in 6-well plates for 48 hours, the medium was replaced with fresh medium every 2 days using a half-dilution method. At each medium replacement, a portion of cells from each group was collected and stained according to the Annexin-V / 7-AAD apoptosis detection kit instructions. The apoptosis rate was continuously detected by flow cytometry. The percentage of early apoptotic (Q3) cells and the percentage of late apoptotic and necrotic (Q2) cells were counted, and the sum of the two percentages was recorded as the apoptosis rate.

[0049] 4. Cell proliferation detection NALM6 and SU-DHL-8 cells were cultured in 96-well plates for 0h, 24h, 48h, and 72h. 10 μL of LCK-8 reagent was added to each well, and the cells were cultured for another 2h. The absorbance (OD value) at 450nm was measured using a microplate reader. Simultaneously, the cell growth status was recorded in real time using the Incucyte® S3 live cell imaging analysis system, and the relative cell proliferation rate was calculated as (OD value of the high-leucine group / OD value of the normal control group × 100%).

[0050] (III) Experimental Results 1. Results of apoptosis detection ( Figure 1 In the high-leucine group (550 mg / L), the apoptosis rate of NALM6 cells reached over 40%, and the apoptosis rate of SU-DHL-8 cells reached over 35%, which were significantly higher than those of the normal control group (apoptosis rate <10%), indicating that leucine can significantly induce apoptosis in hematologic tumor cells.

[0051] 2. Cell proliferation detection: Compared with the normal control group, the OD values ​​of NALM6 and SU-DHL-8 cells in the high-leucine group began to decrease significantly after 24 h, and the relative cell proliferation rate decreased to below 50% at 72 h. Figure 1 (B and D) The experimental results showed that tumor cell growth was significantly inhibited under high leucine conditions, indicating that leucine can effectively inhibit the in vitro proliferation of hematologic tumor cells.

[0052] Example 3: In vivo therapeutic experiment of leucine in animal models of hematologic malignancies (a) Experimental materials 1. Animal source: C57BL / 6 mice were purchased from Shanghai Nanmo Biotechnology Co., Ltd.

[0053] 2. Leucine diet: Regular mouse diet (AIN-93G Growing Rodent Diet, Ready Bite®, with the standard dose of leucine, purchased from Ruide Biotechnology), and high-leucine mouse diet with 5 times the leucine dose (purchased from Ruide Biotechnology, AIN-93G Growing Rodent Diet with 5× Leucine, Ready Bite®, with an additional 66 grams of L-leucine per kilogram of AIN-93G Growing Rodent Diet, while reducing the amount of corn starch added to keep the total calories consistent with AIN-93G Growing Rodent Diet).

[0054] 3. Experimental instruments: fully automated blood analyzer, small animal survival monitoring system.

[0055] (II) Experimental Methods 1. Model establishment: 1×10⁻⁶ C57BL / 6 mice were injected via the tail vein. 6 A mouse model of MH / NRASG12D mutant B-cell precursor acute lymphoblastic leukemia (BCP-ALL) was constructed using MH / NRASG12D BCP-ALL cells (abbreviated as BCP-ALL leukemia mouse model). 2. Dietary intervention: Twenty BCP-ALL leukemia mice, aged 6-8 weeks, half male and half female, were randomly divided into a normal diet group and a high-leucine diet group, with 10 mice in each group. The normal diet group was fed a standard mouse diet, while the high-leucine diet group was fed a high-leucine diet with 5 times the leucine dose. Both groups had free access to food and water, and the intervention continued until the end of the experiment. 3. Peripheral blood testing: Blood was collected from the tail vein of mice every 7 days, and routine blood indicators were tested using a fully automated blood analyzer. The levels of hemoglobin (Hb) and platelets (PLT) were the main parameters to assess anemia and thrombocytopenia in the mice. 4. Survival analysis: The survival time of mice was recorded using a small animal survival monitoring system, Kaplan-Meier survival curves were plotted, and the median survival time was calculated.

[0056] (III) Experimental Results 1. Blood routine indicators: Two weeks after the BCP-ALL leukemia mouse model was fed a standard mouse diet (normal diet group), a significant decrease in hemoglobin and platelet count was observed. Figure 2 In the middle B group, the mice exhibited severe anemia and thrombocytopenia; while the high-leucine diet group showed significantly higher hemoglobin and platelet levels than the normal diet group, and the symptoms of anemia and thrombocytopenia were significantly improved.

[0057] 2. Survival: Compared with the normal diet group (median survival time was 15 days), the median survival time of the high-leucine diet group was significantly prolonged (median survival time was 18 days). Figure 2 (A) indicates that a high-leucine diet can significantly improve the survival of animal models of hematologic malignancies, demonstrating in vivo antitumor and prognostic effects.

[0058] Example 4: Molecular mechanism of leucine's anti-hematologic malignancy (a) Experimental materials 1. Tumor cell lines: The NALM6 cell line, a B-cell precursor acute lymphoblastic leukemia, and the SU-DHL-8 cell line, a B-cell lymphoma, were both purchased from the ATCC cell bank; 2. Leucine: Leucine powder with a purity of ≥98% (Targetmol); 3. Cell culture medium: RPMI-1640 complete medium, prepared as follows: Add 10% fetal bovine serum (Gibco) and 1% penicillin and streptomycin to the RPMI-1640 (Shanghai Yuanpei Biotechnology) basal medium. The penicillin and streptomycin are penicillin and streptomycin respectively. The concentration of penicillin in the RPMI-1640 complete medium is 100 U / mL and the concentration of streptomycin is 100 μg / mL.

[0059] 4. Experimental reagents: Annexin-V / 7-AAD apoptosis detection kit and CCK-8 proliferation detection kit were purchased from Thermo; C75 inhibitor was purchased from Targetmol, CAS No.: 218137-86-1, purity ≥98%, catalog number T10657; RNA extraction kit was purchased from Tiangen Biotech.

[0060] 5. Experimental instruments: Illumina sequencing platform, flow cytometer, clean bench, carbon dioxide incubator.

[0061] (II) Experimental Methods 1. Cell Culture NALM6 and SU-DHL-8 cells were placed in carbon dioxide incubators at 37°C and 5% CO2, respectively, and cultured routinely in RPMI-1640 complete medium. Cells in the logarithmic growth phase were used for experiments.

[0062] 2. Leucine treatment NALM6 cells were treated with leucine as follows: Drug-containing culture media were prepared, with a normal control group (RPMI-1640 complete medium containing leucine at a concentration of 50 mg / L, the standard cell culture concentration) and a high-leucine group (RPMI-1640 complete medium supplemented with leucine to achieve a concentration of 550 mg / L). NALM6 tumor cells in logarithmic growth phase were collected, and the cell density was adjusted to obtain 5 × 10⁶ cells / year. 5 2 mL of cell suspension per well was added to each well of a 6-well plate. After cell seeding, the 6-well plates were set up as a normal control group and a high-leucine group, and the corresponding culture medium was added to each group. Each group was divided into 3 replicates and placed in a 37°C, 5% CO2 incubator.

[0063] 3. RAN extraction and next-generation sequencing When NALM6 cells treated with different concentrations of leucine exhibited obvious apoptotic phenotypes, cells from each group were collected, and total RNA was extracted using an RNA extraction kit. RNA concentration and integrity were then measured. After passing quality control, transcriptome sequencing libraries were constructed, and RNA sequencing (RNA-seq) analysis was performed using the Illumina next-generation high-throughput sequencing platform.

[0064] Differential expression analysis and gene set enrichment analysis (GSEA) were performed on sequencing data to screen signaling pathways and key regulatory genes related to lipid metabolism, de novo fatty acid synthesis, and energy metabolism, in order to elucidate the molecular mechanism by which a high-leucine environment induces apoptosis in leukemia cells.

[0065] 4. Pathway blocking experiment: RNA-seq results showed that lipid de novo synthesis pathways were significantly inhibited after high leucine treatment. Therefore, functional verification experiments were further conducted using the fatty acid synthase (FASN) inhibitor C75.

[0066] NALM6 cells were treated with different concentrations of C75 (20, 30, and 40 μM) to simulate the inhibitory effect of high-concentration leucine on lipid synthesis. After treatment for a certain period, the level of cell apoptosis was detected by Annexin V-FITC / PI double staining combined with flow cytometry, and the relationship between cell apoptosis rate and C75 concentration was analyzed to verify the key role of lipid metabolism pathways in leucine-induced leukemia cell death.

[0067] (III) Experimental Results 1. Transcriptome sequencing results (see...) Figure 5 (A, B1-B3 and C) Transcriptome sequencing results showed that, compared with normal culture conditions, the high-leucine environment significantly downregulated the expression of key genes related to de novo lipid synthesis, including genes involved in fatty acid synthesis and lipid metabolism regulation. Meanwhile, GSEA enrichment analysis indicated that multiple lipid synthesis and fatty acid metabolism pathways were significantly inhibited.

[0068] The above results indicate that high leucine can remodel the lipid metabolism network of leukemia cells, inhibit de novo lipid synthesis and energy metabolism processes, thereby affecting the survival status of tumor cells and promoting their apoptosis.

[0069] 2. Results of lipid metabolism pathway blocking experiments ( Figure 5 (D1-D2) Treatment of NALM6 cells with the fatty acid synthesis inhibitor C75 significantly increased the apoptosis rate, exhibiting a concentration-dependent trend. Flow cytometry analysis revealed a significant increase in both early and late apoptotic cell proportions with increasing C75 concentration.

[0070] The above results further demonstrate that the de novo lipid synthesis pathway plays an important role in the survival of leukemia cells; leucine may induce programmed cell death in leukemia cells by inhibiting lipid synthesis and metabolism pathways.

[0071] Example 5: Clinical application of leucine in patients with hematologic malignancies (a) Target audience 1. Patients with non-Philadelphia B-cell acute lymphoblastic leukemia (non-Ph B-ALL); 2. B-cell lymphoma patients receiving CAR-T cell therapy; 3. Patients with other malignant lymphomas such as Hodgkin's lymphoma and diffuse non-Hodgkin's lymphoma.

[0072] Leucine supplementation intervention is mainly applicable to patients in the above three types of patients whose baseline plasma leucine level is lower than the median of the normal reference range (the normal reference range for adult plasma leucine is 55–205 μmol / L), lower than the average level of healthy controls, or located in the lowest 25th percentile of the target population.

[0073] (ii) Excluding the crowd 1. Severe liver dysfunction: ALT or AST > 3 times the upper limit of normal; 2. Severe renal insufficiency: eGFR < 30 mL / min / 1.73m²; 3. Accompanied by severe metabolic diseases: such as syrup maple syrup disease and other branched-chain amino acid metabolism disorders; 4. Individuals with uncontrolled active infections; 5. Pregnant or breastfeeding women; 6. Individuals allergic to amino acid preparations.

[0074] (III) Leucine intervention methods Leucine supplement: Leucine powder, 5g / sachet. Adults take one sachet twice daily, dissolved in warm water.

[0075] The formula composition of leucine powder is: 95 parts by weight of leucine and 5 parts by weight of mannitol (as filler and flavoring agent), with a total weight of 5g per bag, including 4.75g of leucine.

[0076] Preparation method of leucine powder: Mix leucine powder and mannitol powder in a certain proportion, place them in a mixer and mix thoroughly for 30 minutes. After mixing evenly, package them into 5g bags using a powder dispensing machine, and seal them to obtain leucine powder. This powder is easily soluble in water, has a mild taste, no obvious odor, and is convenient for patients to take orally. The shelf life is 24 months at room temperature.

[0077] Plasma leucine levels were detected using high performance liquid chromatography (HPLC), and leucine intake was dynamically adjusted based on the patient's age and baseline metabolic status to maintain plasma leucine concentration in the middle to high range of the normal reference range for the corresponding age, preferably near the upper limit of the normal reference range. For example, if the normal reference range for adult plasma leucine is 55–205 μmol / L, then the plasma leucine level should be maintained at 130–175 μmol / L.

Claims

1. The application of leucine in the preparation of drugs that inhibit the proliferation of hematologic malignancies and induce apoptosis in hematologic malignancies, characterized in that... The hematologic malignancies include lymphoid malignancies, specifically B-cell precursor acute lymphoblastic leukemia, B-cell lymphoma, Hodgkin lymphoma, and non-Hodgkin lymphoma.

2. The application of leucine in the preparation of drugs to improve the clinical prognosis of patients with hematologic malignancies, characterized in that... The drug is indicated for patients with non-Philadelphia B-cell acute lymphoblastic leukemia and patients with B-cell lymphoma receiving CAR-T cell therapy.

3. Application of leucine in the preparation of adjuvant drugs for CAR-T cell therapy of hematologic malignancies.

4. The application according to any one of claims 1-3, characterized in that... The drug uses leucine as its core active ingredient and can be composed of leucine alone or in combination with edible excipients. Dosage forms include powder, granules, capsules, and oral liquid.

5. The application according to claim 4, characterized in that... The drug contains 90-98 parts by weight of leucine and 5 parts by weight of mannitol.

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