Application of IL-1alpha inhibitor in preparation of medicine for treating cardiac injury induced by leukemia treatment

Abstract

The invention discloses application of an IL-1alpha inhibitor in preparation of a medicine for treating heart injury induced by leukemia treatment. The application provided by the invention can effectively reduce cardiovascular complications in the leukemia treatment period, reduce the occurrence rate of long-term cardiovascular adverse events, contribute to improvement of cardiac metabolic disorders and cardiac dysfunction after treatment, possibly and remarkably improve long-term prognosis of leukemia patients, make numerous leukemia patients benefit, and has a wide application prospect. Therefore, obvious social and economic effects are obtained.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of an IL-1α inhibitor in the preparation of a medicine for treating cardiac damage induced by leukemia treatment. Background Art

[0002] Tumor diseases and cardiovascular diseases are two major types of diseases that pose a serious threat to human health. Leukemia accounts for one-third of childhood malignant tumor patients and is the most common childhood malignant tumor. Data show that with the emergence of new treatments and the optimization of treatment plans, the five-year overall survival rates of childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) have reached 90% and 78.2%, respectively, which has continuously expanded the base of leukemia survivors. A recent clinical study showed that the long-term disease burden of leukemia survivors among childhood tumor diseases is much higher than that of survivors of other types of tumors, and the burden is mainly caused by cardiovascular diseases. 1 The incidence of heart disease in children with AML is significantly higher than that in children with ALL, mainly manifested as cardiomyopathy and heart failure. 2 . It is generally believed that the risk of death from heart disease in leukemia survivors is mainly due to tumor chemotherapy. For example, in the 1970s, a study reported that anthracycline chemotherapy drugs, the core drugs in childhood leukemia chemotherapy, have clear dose-dependent cardiac toxicity. 3 A study on childhood AML survivors showed that 12% of survivors will develop cardiac dysfunction within 5 years, of which about 70% will develop during the peri-chemotherapy period. It is particularly important that whether treatment-related cardiac toxicity occurs in the early stage determines the long-term survival rate. 4 Therefore, early identification of chemotherapy-induced cardiotoxicity and active intervention may reduce the burden of cardiovascular disease. 5 .

[0003] Energy metabolism is the most important physiological activity of the heart. Many studies have found that abnormal energy metabolism is an important cause of heart damage. Neubauer et al. first proposed the concept that a heart with heart failure is a "fuel-depleted machine" 6 However, it is still unclear whether chemotherapy drugs can damage the heart by interfering with myocardial energy metabolism. On the other hand, previous studies in the field of oncology cardiology mainly used healthy mice to observe the direct damage of drugs to the heart after long-term, high-dose, multi-course chemotherapy. For example, Wallace et al. found that anthracyclines can damage myocardial cells by promoting the generation of reactive oxygen species in the mitochondrial respiratory chain. 6 Zhang et al. found that anthracyclines can cause cardiac damage by inhibiting the activity of topoisomerase IIβ 7However, clinically, the inventors found that children with leukemia can develop chemotherapy drug-related heart damage during the short-term induction chemotherapy phase. In addition, previous studies have overlooked the indirect toxic effects of changes in the tumor microenvironment on the heart after chemotherapy and ignored the possible interaction among the tumor, chemotherapy drugs, and the heart. These problems have prompted the inventors to consider whether there is an unexplored mechanism of chemotherapy-related heart damage, and then to reduce the burden of cardiovascular diseases in children with leukemia after chemotherapy through precise intervention. Summary of the Invention

[0004] To address the deficiency in the prior art of lacking a precise and effective method for improving heart damage caused by leukemia treatment, such as cardiac metabolic disorders and abnormal cardiac function, the present invention provides the use of an inhibitor of IL-1α in the preparation of a drug for treating heart damage induced by leukemia treatment.

[0005] One of the technical solutions provided by the present invention to solve the above technical problems is: the use of an inhibitor of IL-1α (i.e., Interleukin 1α) in the preparation of a drug for treating heart damage induced by leukemia treatment.

[0006] In a specific embodiment of the present invention, the method of leukemia treatment is selected from one or more of the following: chemotherapy, radiotherapy, molecular targeted therapy, and immunotherapy.

[0007] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0008] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0009] In a specific embodiment of the present invention, the leukemia treatment causes necrosis of leukemia cells.

[0010] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

[0011] In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

[0012] In a specific embodiment of the present invention, the inhibitor of IL-1α is an anti-IL-1α antibody.

[0013] In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

[0014] In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

[0015] In a specific embodiment of the present invention, the anti-IL-1α antibody is purchased from R&D Systems with a product number of AB-400-NA.

[0016] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

[0017] In a specific embodiment of the present invention, the acute lymphocytic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0018] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0019] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0020] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0021] In order to solve the above technical problems, the second technical solution provided by the present invention is: use of an antibody purchased from R&D Systems with a product number of AB-400-NA in the preparation of a drug for treating cardiac damage induced by leukemia treatment.

[0022] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and / or, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia.

[0023] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0024] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0025] In a specific embodiment of the present invention, the acute lymphocytic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0026] In a specific embodiment of the invention, the leukemia treatment results in necrosis of leukemia cells.

[0027] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and / or, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0028] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0029] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0030] To solve the above technical problems, the third technical solution provided by the present invention is: use of an antibody purchased from R&D Systems with a product number of AB-400-NA in the preparation of a drug for treating cardiac damage induced by leukemia treatment by inhibiting IL-1α.

[0031] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and / or, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia.

[0032] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0033] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0034] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0035] In a specific embodiment of the invention, the leukemia treatment results in necrosis of leukemia cells.

[0036] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and / or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0037] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0038] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0039] To solve the above technical problems, the fourth technical solution provided by the present invention is: use of an antagonist of IL1R1 (i.e., interleukin-1 receptor type I) in the preparation of a drug for treating cardiac damage induced by leukemia treatment.

[0040] The present invention has demonstrated that knockout of cardiac IL1R1 receptor can achieve a protective effect on cardiac function (Example 2). Based on this, those skilled in the art can reasonably expect that IL1R1 antagonists can treat leukemia-induced cardiac damage.

[0041] In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

[0042] In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

[0043] In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra (Anakinra, a recombinant, non-glycosylated form of human interleukin-1 receptor antagonist (IL-1Ra), CAS No.: 143090-92-0).

[0044] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

[0045] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0046] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and / or, the leukemia treatment causes leukemia cell necrosis.

[0047] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and / or the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0048] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0049] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and / or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0050] To solve the above technical problems, the fifth technical solution provided by the present invention is: a method for treating cardiac damage induced by leukemia treatment, the method comprising administering a therapeutically effective amount of an IL-1α inhibitor to a patient in need.

[0051] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

[0052] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0053] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the leukemia treatment results in necrosis of leukemia cells.

[0054] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

[0055] In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

[0056] In a specific embodiment of the present invention, the IL-1α inhibitor is an anti-IL-1α antibody.

[0057] In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

[0058] In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

[0059] In a specific embodiment of the present invention, the anti-IL-1α antibody is purchased from R&D Systems with a product number of AB-400-NA.

[0060] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

[0061] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0062] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0063] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0064] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0065] In a specific embodiment of the invention, the patient is a child.

[0066] To solve the above technical problems, the sixth technical solution provided by the present invention is: a method for treating cardiac damage induced by leukemia treatment, the method comprising administering a therapeutically effective amount of an antibody purchased from R&D Systems with a product number of AB-400-NA to a patient in need.

[0067] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and / or, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia.

[0068] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0069] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0070] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0071] In a specific embodiment of the invention, the leukemia treatment results in necrosis of leukemia cells.

[0072] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and / or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0073] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0074] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0075] In a specific embodiment of the invention, the patient is a child.

[0076] In order to solve the above technical problems, the seventh technical solution provided by the present invention is: a method for treating cardiac damage induced by leukemia treatment, the method comprising administering a therapeutically effective amount of an antagonist of IL1R1 to a patient in need.

[0077] In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

[0078] In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

[0079] In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra.

[0080] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

[0081] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0082] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy. In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and / or, the leukemia treatment causes necrosis of leukemia cells.

[0083] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and / or the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0084] In a specific embodiment of the present invention, the acute lymphocytic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0085] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and / or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0086] In a specific embodiment of the invention, the patient is a child.

[0087] As used herein, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal or human that is sought, for example, by a researcher or clinician. In addition, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that is effective to enhance normal physiological function.

[0088] In order to solve the above technical problems, the eighth technical solution provided by the present invention is: an inhibitor of IL-1α, which is used for treating cardiac damage induced by leukemia treatment.

[0089] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

[0090] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0091] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0092] In a specific embodiment of the invention, the leukemia treatment results in necrosis of leukemia cells.

[0093] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug.

[0094] In a specific embodiment of the present invention, the anthracycline chemotherapy drug is daunorubicin.

[0095] In a specific embodiment of the present invention, the IL-1α inhibitor is an anti-IL-1α antibody.

[0096] In a specific embodiment of the present invention, the anti-IL-1α antibody is a monoclonal antibody.

[0097] In a specific embodiment of the present invention, the anti-IL-1α antibody is a neutralizing antibody.

[0098] In a specific embodiment of the present invention, the anti-IL-1α antibody is purchased from R&D Systems with a product number of AB-400-NA.

[0099] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphoblastic leukemia.

[0100] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0101] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0102] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0103] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0104] To solve the above technical problems, the ninth technical solution provided by the present invention is: an antibody purchased from R&D Systems with the product number AB-400-NA, which is used to treat cardiac damage induced by leukemia treatment.

[0105] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and / or, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia.

[0106] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0107] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0108] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0109] In a specific embodiment of the invention, the leukemia treatment results in necrosis of leukemia cells.

[0110] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and / or the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

[0111] In a specific embodiment of the present invention, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0112] In a specific embodiment of the present invention, the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0113] In order to solve the above technical problems, the tenth technical solution provided by the present invention is: an antagonist of IL1R1, which is used for treating cardiac damage induced by leukemia treatment.

[0114] In a specific embodiment of the present invention, the antagonist of IL1R1 is an anti-IL1R1 drug.

[0115] In a specific embodiment of the present invention, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1.

[0116] In a specific embodiment of the present invention, the competitive inhibitory drug is Anakinra.

[0117] In a specific embodiment of the present invention, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy.

[0118] In a specific embodiment of the present invention, the immunotherapy is CAR-T therapy.

[0119] In a specific embodiment of the present invention, the immunotherapy is CD19 CAR-T therapy.

[0120] In a specific embodiment of the present invention, the chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; and / or, the leukemia treatment causes necrosis of leukemia cells.

[0121] In a specific embodiment of the present invention, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia; and / or the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure.

[0122] In a specific embodiment of the present invention, the acute lymphoblastic leukemia is acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia.

[0123] In a specific embodiment of the present invention, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and / or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

[0124] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0125] The reagents and raw materials used in the present invention are commercially available.

[0126] The positive and progressive effects of the present invention are:

[0127] The present invention first proposes that the release of tumor-derived cytokine IL-1α caused by leukemia treatment (such as chemotherapy or immunotherapy) is the key to causing cardiac energy metabolism remodeling and dysfunction, emphasizing that the interaction between drugs and tumors plays an important role in the cardiotoxicity of leukemia treatment drugs (such as chemotherapy drugs or CAR-T). The use of the IL-1α inhibitor provided by the present invention in the preparation of drugs for treating leukemia treatment-induced cardiac damage will effectively reduce cardiovascular complications during leukemia treatment, reduce the incidence of long-term adverse cardiovascular events, and help improve cardiac metabolic disorders and abnormal cardiac function after treatment. It is likely to significantly improve the long-term prognosis of leukemia patients, benefiting many leukemia patients, thereby achieving significant social and economic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] Figure 1Construction of cardiac-specific Il1r1 knockout mice. (A) Construction diagram; (B) Immunoblotting experiment to verify the IL1R1 knockout effect, the control group was IL1R1 fl / fl, the experimental group was IL1R1 cKO, and the right side is the protein expression of IL1R1 relative to Actin. IL1R1: interleukin-1 receptor type 1; Actin: actin. The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( * P<0.05).

[0129] Figure 2 This is an AML cell construction with stable knockdown of Il1a. (A) Construction diagram; (B) RT-PCR verification of the knockout effect. The control group used the scramble sequence and the experimental group used the shIL-1α sequence. The right side shows the mRNA expression of Il1a relative to Gapdh. The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( *** P<0.001).

[0130] Figure 3 To verify the effect of myocardial-specific overexpression of PGC-1α. The control group was AAV9-NC, and the experimental group was AAV9-PGC1α. The right side shows the protein expression of PGC-1α relative to Actin. NC: negative control, PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1-α, Actin: actin. The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( ** P<0.01).

[0131] Figure 4 p65 overexpression plasmid and PGC-1α promoter fluorescent reporter plasmid.

[0132] Figure 5 Schematic diagram of the construction of the MLL-AF9 AML mouse model. AML: acute myeloid leukemia.

[0133] Figure 6The changes in cardiac metabolism and function after DNR chemotherapy in AML mice. (A) AML mice were divided into two groups, one group received DNR chemotherapy (AML+DNR group), and the other group used PBS as a control (AML group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Cardiac PET-CT detection of the two groups of mice. The signal strength of FTHA and FDG probes labeled with fluorine 18 reflects the level of cardiac utilization of fatty acids and glucose, respectively; (D) Myocardial ATP content evaluation of the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; FDG: deoxyglucose; FTHA: 6-thio-heptadecanoic acid; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( * P < 0.05; ** P<0.01 *** P<0.001).

[0134] Figure 7 The expression of key enzymes of cardiac lipid metabolism decreased in leukemia mice after chemotherapy. AML: acute myeloid leukemia; DNR: daunorubicin; CD36: leukocyte differentiation antigen 36; CPT1: carnitine palmitoyltransferase 1; LCAD: long-chain acyl-CoA dehydrogenase; MCAD: medium-chain acyl-CoA dehydrogenase; Actin: actin; The statistical differences between the two groups of samples were calculated using two independent sample t-tests (*P<0.05; **P<0.01; ***P<0.001).

[0135] Figure 8 Single-cell sequencing analysis of myocardium after DNR chemotherapy and normal myocardium in children with AML. (A) UMAP diagram showing the clustering distribution of cardiac cells; (B) scMetabolism metabolic scores of normal myocardium and myocardium after chemotherapy; the sugar metabolism pathway is highlighted in red, and the fatty acid metabolism pathway is highlighted in blue; (C) Pseudo-time analysis of all myocardial cells; (D) Pseudo-time branch point analysis of all myocardial cells. The names of genes related to fatty acid metabolism that are downregulated in non-adaptive myocardial cells are highlighted in blue, and the names of genes related to sugar metabolism that are upregulated are highlighted in red.

[0136] Fig. 9Cardiac metabolic and functional changes in normal tumor-free mice after DNR chemotherapy. (A) Healthy tumor-free mice were divided into two groups, one group received DNR chemotherapy (TF+DNR group), and the other group used PBS (TF group) as a control; (B) Cardiac ultrasound detection of the two groups of mice; (C) Cardiac PET-CT detection of the two groups of mice; (D) Myocardial ATP content detection of the two groups of mice; TF: tumor-free; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; FTHA: 6-thio-heptadecanoic acid; FDG: deoxyglucose; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-test (NS: no significant difference).

[0137] Fig.10 This is a transcriptomic analysis of the heart of AML mice after DNR chemotherapy. (A) KEGG pathway enrichment analysis of differentially expressed genes in cardiac transcriptomic results after chemotherapy, with cytokine and cytokine interaction pathways significantly enriched and highlighted in red; (B) Biological response pathway analysis of cardiac transcriptomic results after chemotherapy, with PPAR signaling significantly downregulated and highlighted in blue; (C) Protein interaction network analysis of differentially expressed genes in the PPAR pathway, showing that IL-1 signaling, NF-KappaB signaling and the key metabolic protein PPARGC1A are closely interacting.

[0138] Fig.11 Plasma multifactor detection after DNR chemotherapy in AML mice, the red box indicates IL-1α. TF: tumor-free; AML: acute myeloid leukemia; DNR: daunorubicin. The differences between samples were calculated using ANOVA post hoc Tukey-Kramer test (NS: no significant difference; *P<0.05; **P<0.01; ***P<0.001).

[0139] Fig.12 IL-1α is released by necrosis of AML cells after DNR chemotherapy. (A) Flow cytometry was used to detect the ratio of live cells to necrotic cells in AML primary cell lines after 24 hours of intervention with different DNR concentration gradients; (B) Detection of IL-1α content in the cell culture supernatant after 24 hours of intervention with 0.5μM DNR in AML primary cell lines. IL-1α: interleukin 1α; DNR: daunorubicin; The statistical difference between the two groups of samples was calculated using the two-independent sample t-test, and the difference between more than two groups of samples was calculated using the post hoc Tukey-Kramer test for analysis of variance (*P<0.05; **P<0.01; ***P<0.001).

[0140] Fig.13Cardiac metabolic and functional changes after IL-1α administration in normal tumor-free mice. (A) Healthy tumor-free mice were divided into two groups, one group received IL-1α (TF+IL-1α group), and the other group used PBS as a control (TF group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Cardiac PET-CT detection of the two groups of mice; (D) Myocardial ATP content detection of the two groups of mice. TF: tumor-free; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction fraction; FTHA: 6-thio-heptadecanoic acid; FDG: deoxyglucose; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( * P < 0.05; ** P<0.01 *** P<0.001).

[0141] Fig.14 Cardiac-specific Il1r1 knockout improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention in AML mice with cardiomyocyte-specific IL1R1 knockout (IL1R1 cKO group) and control AML mice (IL1R1 fl / fl group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests (***P<0.001).

[0142] Fig.15 To analyze the correlation between the increase rate of plasma IL-1α and the decrease rate of LVEF in patients with acute myeloid leukemia before and after chemotherapy. LVEF: left ventricular ejection fraction.

[0143] Fig.16 Knockdown of Il1a in AML cells improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention using control (scramble group) and IL-1α knockdown (shIL-1α group) cells to construct AML models; (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. WT: wild type; AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( *** P<0.001).

[0144] Fig.17Detection of cardiac NF-KappaB signaling pathway in normal tumor-free mice and AML mice after DNR chemotherapy. (A) Immunoblotting detection of cardiac NF-KappaB pathway in normal tumor-free mice after chemotherapy; (B) Immunoblotting detection of cardiac NF-KappaB pathway in AML mice after chemotherapy. TF: tumor-free; AML: acute myeloid leukemia; DNR: daunorubicin; p-p65: phosphorylated transcription factor p65; p65: transcription factor p65; IκBα: nuclear factor κB inhibitor protein; Actin: actin. The statistical difference between the two groups of samples was calculated using two independent sample t-tests (NS: not significant difference; * P < 0.05; ** P<0.01).

[0145] Fig.18 Immunofluorescence and immunoblotting detection of the myocardium of AML children after DNR chemotherapy and normal myocardium. (A) Immunofluorescence detection of IL-1α in the myocardium after chemotherapy (Patient) and normal myocardium (Control); gray indicates ACTIN2, green indicates IL-1α, and blue indicates DAPI; scale: 50μm; (B) Immunoblotting detection of NF-KappaB signaling pathway in the myocardium after chemotherapy and normal myocardium. PGC-1α: peroxisome proliferator receptor γ coactivator 1-α; p65: transcription factor p65; p-p65: phosphorylated transcription factor p65; Actin: actin.

[0146] Fig.19 NF-KappaB signaling antagonist improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention in AML mice treated with PDTC (PDTC group) or not treated (control group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; PDTC: ammonium pyrrolidine dithiocarbamate; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests (**P<0.01; ***P<0.001).

[0147] Fig. 20The expression changes of PGC-1α in the heart of AML mice after DNR chemotherapy. (A) Detection of PGC-1α protein expression in the heart of AML mice after chemotherapy. The right side shows the protein expression of PGC-1α relative to Actin; (B) Detection of PGC-1α transcription level in the heart of AML mice after chemotherapy. AML: acute myeloid leukemia; DNR: daunorubicin; PGC-1α: peroxisome proliferator receptor γ coactivator 1-α; Actin: actin. The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( ** P<0.01).

[0148] Fig.21 Expression of myocardial PGC-1α in children with AML after DNR chemotherapy. (A) UMAP image shows that PGC-1α is mainly expressed in cardiomyocytes in cardiac tissue; (B) Pseudo-time series analysis shows the cell fate of different cardiomyocyte subpopulations; (C) PGC-1α expression levels in different cardiomyocyte subpopulations; (D) Pseudo-time series analysis shows the relationship between different cardiomyocyte fates and PGC-1α expression levels.

[0149] Fig. 22 This is a dual luciferase reporter assay. Overexpression of p65 significantly inhibited the transcription of PGC-1α. TSS: transcription start site; LUC: firefly enzyme; RLU: relative fluorescence intensity; PGC-1α: peroxisome proliferator-activated receptor γ coactivator 1-α; p65: transcription factor p65 ( *** P<0.001).

[0150] Fig.23 Overexpression of PGC-1α in the heart improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention in AML mice with overexpression of PGC-1α in the heart (AAV9-PGC1α group) and control AML mice (AAV9-NC group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. WT: wild type; AAV9: adeno-associated virus type 9; NC: negative control; PGC-1α: peroxisome proliferator receptor gamma coactivator 1-α; AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests ( * P < 0.05; *** P<0.001).

[0151] Fig.24IL-1α neutralizing antibody improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram of DNR intervention in AML mice treated with IL-1α neutralizing antibody (IL-1αAb group) or isotype control antibody (IgG group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice; (D) The proportion of residual GFP+ tumor cells in the bone marrow of the two groups of mice on the 4th day after the end of chemotherapy. AML: acute myeloid leukemia; DNR: daunorubicin; Ab: antibody; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; BM: bone marrow; GFP: green fluorescent protein; The statistical difference between the two groups of samples was calculated using two independent sample t-tests (NS: no significant difference; * P < 0.05; ** P<0.01).

[0152] Fig.25 This is a diagram of the mechanism of chemotherapy-induced cardiac metabolic disorders and functional damage. After chemotherapy, tumor cell necrosis releases IL-1α, which acts on the cardiac IL1R1 receptor to activate the NF-KappaB signaling pathway and inhibit the expression of PGC-1α, leading to cardiac metabolic disorders and functional abnormalities.

[0153] Fig.26 IL-1α neutralizing antibody improves cardiac metabolism and function in T-ALL mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention in T-ALL mice treated with IL-1α neutralizing antibody (IL-1αAb group) or isotype control antibody (IgG group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. T-ALL: acute lymphoblastic T-cell leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical differences between the two groups of samples were calculated using two independent sample t-tests (**P<0.01; ***P<0.001).

[0154] Fig. 27IL-1α neutralizing antibody improves cardiac metabolism and function in B-ALL mice after CAR-T treatment. (A) Experimental schematic diagram, CD19 CAR-T cells intervened in B-ALL mice treated with IL-1α neutralizing antibody (IL-1αAb group) or isotype control antibody (IgG group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. B-ALL: acute lymphoblastic B-cell leukemia; CAR-T: chimeric antigen receptor T cell immunotherapy; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical differences between the two groups of samples were calculated using two independent sample t-tests (*P<0.05; **P<0.01).

[0155] Fig.28 Anakinra improves cardiac metabolism and function in mice after chemotherapy. (A) Experimental schematic diagram, DNR intervention in AML mice treated with Anakinra (Anakinra group) or not treated (control group); (B) Cardiac ultrasound detection of the two groups of mice; (C) Myocardial ATP content detection of the two groups of mice. AML: acute myeloid leukemia; DNR: daunorubicin; LVEF: left ventricular ejection fraction; LVFS: left ventricular short-axis contraction rate; ATP: adenosine triphosphate; The statistical difference between the two groups of samples was calculated using two independent sample t-tests (***P<0.001). DETAILED DESCRIPTION

[0156] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0157] Experimental methods

[0158] Mouse cardiac function assessment

[0159] Fujifilm Vevo3100 small animal ultrasound can achieve high-resolution imaging of the heart. The present invention uses ultrasound to evaluate the cardiac function of leukemia mice before and after chemotherapy, and clarifies the changes in cardiac function caused by leukemia chemotherapy. More accurate cardiac function data is obtained through the 9.4T Bruker BioSpin small animal magnetic resonance, which is equipped with a mouse-specific heart coil and a heart gating device, which can achieve accurate evaluation of mouse cardiac function.

[0160] Cardiac isotope tracer metabolic flux detection

[0161] Cardiac isotope tracer metabolic flux detection can accurately reflect the specific flow direction and flux of glycolipid metabolic substrates in cardiac energy metabolism. The present invention uses C13-labeled glucose and palmitic acid from Sigma, and selects a C13-glucose injection amount of 1 mg / g according to the method reported by Motoaki Sano et al. (J Mol Cell Cardiol. 2015 May; 82: 116-24.), and a C13-palmitic acid injection amount of 0.5 mg / g is selected according to the BSA heating dissolution method reported by Kim et al. (Sci Rep. 2017; 7: 4335.) and the tolerance of mice. The mouse heart is removed 15 minutes after injection for subsequent mass spectrometry analysis.

[0162] Preparatory Example

[0163] Construction of Leukemia Mouse Therapeutic Model

[0164] AML mouse model construction:

[0165] The leukemia mouse chemotherapy model used in the present invention is based on the transfection of primary myeloid stem / progenitor cells of normal mice with lentivirus-packaged MLL-AF9 plasmids, and then GFP+ leukemia cells are screened by flow sorting technology, and further transplanted into mice whose myeloid cells are eliminated after irradiation for amplification, thereby constructing MLL-AF9 AML mice. This model retains the immune function of the recipient mice, can simulate the pathophysiological process of childhood leukemia, and is a currently recognized AML model in the field of hematological tumors. Mice that receive leukemia cell bone marrow transplants will develop leukemia symptoms such as splenomegaly and fatigue after about 20-30 days.

[0166] Construction of T-ALL mouse model (reference: PD-1 signaling defines and protects leukaemic stem cells from T cell receptor-induced cell death in T cell acute lymphoblastic leukaemia. DOI: 10.1038 / s41556-022-01050-3):

[0167] The T-ALL mouse model used is based on transfection of primary myeloid stem / progenitor cells of normal mice with lentiviral packaging of NOTCH1 plasmid, followed by selection of GFP+ leukemia cells by flow cytometry, and further transplantation into irradiated mice for expansion to construct T-ALL mice. Overactivation mutations of NOTCH1 occur in more than 60% of human T-ALL.

[0168] Construction of B-ALL mouse model (reference: Ink4a and Arf are crucial factors in the determination of the cell of origin and the therapeutic sensitivity of Myc-induced mouse lymphoid tumor. DOI: 10.1038 / onc.2011.462):

[0169] The B-ALL mouse model used is based on transfection of primary hematopoietic stem cells of normal mice with lentivirus-packaged N-Myc plasmid, followed by selection of GFP+ leukemia cells by flow cytometry, which are then transplanted into irradiated mice for expansion, thereby constructing B-ALL mice.

[0170] The chemotherapy regimen used in the present invention is based on the anthracycline dosage of clinical leukemia children in the induction chemotherapy stage, and adopts 5 mg / kg of DNR for two consecutive days for chemotherapy.

[0171] The CAR-T treatment regimen used in the present invention (reference: Therapeutic efficacy of anti-CD19 CAR-T cells in a mouse model of systemic lupus erythematosus. DOI: 10.1038 / s41423-020-0472-1):

[0172] 1. Composition of chimeric antigen receptor: The variable regions of the light and heavy chains of the 1D3 anti-CD19 antibody were used as scFv fragments, mCD8 was used as the transmembrane region, and m4-1BB and mCD3ζ signals were used as the cytoplasmic region. The above sequences were synthesized and connected to a lentiviral vector with an IRES-EGFP fluorescent signal.

[0173] 2. After lentivirus infects T cells, CD19 CAR-T cells are obtained.

[0174] 3. 1E6 CAR-T cells were given to each mouse for two consecutive days (B-ALL mice were cleared of lymph nodes).

[0175] Construction of cardiac-specific Il1r1 knockout mice and systemic Il1a knockout mice

[0176] Il1r1-Flox mice were purchased from Shanghai Model Organisms Technology Co., Ltd. The flox site was inserted into the 3-4 exon region of the Il1r1 gene in this strain of mice, and then the Il1r1 was constructed by mating with Myh6-CreERT2 transgenic mice. flox / flox ; Myh6-CreERT2 mice. After 5 days of intervention with 100 mg / kg tamoxifen and 3 weeks of waiting, immunoblotting showed that the expression of IL1R1 protein in the myocardial tissue of mice was significantly reduced, thus confirming the effectiveness of this genetically engineered mouse strain ( Figure 1 ). Systemic Il1a knockout mice were purchased from Shanghai Model Organisms Technology Co., Ltd. This strain of mice had the exon 5-6 region of the Il1a gene knocked out, and the genotype had been verified by sequencing.

[0177] Construction of AML cells with Il1a knockdown

[0178] Molecular cloning technology was used to modify the commercialized MISSION shRNA plasmid of Sigma, adding the red fluorescent protein gene sequence (RFP) and the shRNA sequence targeting mouse Il1a. The plasmid was then packaged with lentivirus and transfected into primary AML cells, and finally GFP+RFP+ cells were sorted by flow cytometry. RT-PCR experiments verified that the expression of Il1a in GFP+RFP+ AML cells was significantly reduced, proving that an AML cell line with stable knockdown of Il1a was successfully constructed ( Figure 2 ).

[0179] AAV9-PGC-1α virus construction

[0180] AAV9-PGC-1α virus carrying a myocardial specific promoter was purchased from Shanghai Hanheng Biotechnology Co., Ltd. In the present invention, the applicant used tail vein injection of the virus, and the number of viruses was selected as 4*10 13 vg / kg injection (Gene Ther. 2011 Jan; 18(1): 43-52). Three weeks after wild-type mice were injected with the virus, immunoblotting confirmed that the expression of PGC-1α in their hearts was significantly increased ( Figure 3 ).

[0181] Construction of p65 overexpression plasmid and PGC-1α promoter fluorescence reporter plasmid

[0182] The present invention cloned the amplified human p65 full-length coding sequence (NM_021975) into the CV702 vector, and simultaneously cloned the human PGC-1α (NM_013261) promoter sequence covering a 1100 bp fragment (from -1000 bp to +100 bp) into the GV238 luciferase vector ( Figure 4 ).

[0183] Example 1 Leukemia chemotherapy induces cardiac energy metabolism disorder and heart damage

[0184] The heart is an organ with a high energy demand. It beats more than 100,000 times a day, transports 10 tons of blood to the whole body, and consumes 6 kilograms of ATP. 6 In the fasting state, 70% of the heart's energy is supplied by fatty acids, so energy conversion is the most core metabolic process of the heart. Heart energy metabolism in heart failure will be remodeled, from fatty acid energy supply to glucose energy supply, accompanied by decreased cardiac ATP synthesis and dysfunction. 8 Many studies have confirmed the close relationship between cardiac energy metabolism disorders and heart damage. For example, in a recent study, the applicant found that energy imbalance induced by insulin resistance in adolescence will directly lead to heart damage and dysfunction. 9 However, is the cardiac toxicity in children with leukemia after chemotherapy related to abnormal energy metabolism? Can the cardiovascular damage caused by chemotherapy be alleviated by improving cardiac energy metabolism? These questions are still unclear and need further exploration.

[0185] It is generally believed that gene fusion is the main pathogenesis of childhood leukemia 10 MLL-AF9 fusion protein is present in 25% of newly diagnosed AML in children 11 The AML mouse model carrying this fusion protein is widely used in the field of childhood blood tumor research. The inventors transfected the MLL-AF9 fusion gene into mouse myeloid cells using retrovirus and constructed an acute myeloid leukemia (MLL-AF9 AML) mouse tumor model ( Figure 5 Then, the inventors divided the model mice into two groups after they showed signs of leukemia. One group used the anthracycline chemotherapy drug daunorubicin (DNR) for intervention (5 mg / kg of DNR was given for two consecutive days) according to the clinical AML short-term induction chemotherapy regimen, and the other group used phosphate buffered saline (PBS) as a control ( Figure 6 The inventors further used small animal cardiac ultrasound to detect the changes in left ventricular ejection fraction (LVEF) and left ventricular short-axis contraction fraction (LVFS) in mice after chemotherapy. The results showed that compared with the control group, the cardiac function of leukemia mice after short-term induction chemotherapy was significantly decreased ( Figure 6 Then, the inventors used PET-CT to evaluate the changes in cardiac energy metabolism in leukemia mice after chemotherapy. The results showed that compared with the control group, the heart of leukemia mice after chemotherapy had reduced fatty acid uptake and enhanced glucose uptake ( Figure 6 Further, the inventors detected the ATP content in the myocardial tissue of leukemia mice after chemotherapy, and the results showed that the ATP content in the heart decreased significantly after chemotherapy ( Figure 6 Finally, the inventors used immunoblotting to evaluate the expression of lipid metabolism-related proteins, and the results showed that the expression of key enzymes of cardiac lipid metabolism was significantly reduced after chemotherapy ( Figure 7 ). These results demonstrate that leukemia mice had impaired fatty acid metabolism after chemotherapy, accompanied by decreased cardiac function. To further verify the inventors’ conclusions in human specimens, the inventors collected myocardial biopsy specimens from AML children with abnormal cardiac function after DNR chemotherapy and normal myocardial specimens from children who had been brain dead, and performed single-cell transcriptome analysis. The results also showed that myocardial fatty acid metabolism decreased and glucose metabolism increased in children after chemotherapy ( Figure 8 ). In summary, the inventors believe that cardiac energy metabolism remodeling is closely related to chemotherapy-induced cardiac damage and decreased cardiac function, and impaired fatty acid metabolism is an important cause of cardiac dysfunction.

[0186] Example 2 Tumor cell-derived IL-1α causes cardiac metabolic and functional disorders

[0187] What factors drive the changes in cardiac energy metabolism in leukemia mice after chemotherapy? Studies have shown that in vitro perfusion of anthracyclines in the heart reduces carnitine content and thus affects cardiac metabolism 12 Drugs approved for the treatment of metabolic diseases such as diabetes, such as metformin (MET) and empagliflozin (EMPA), can prevent anthracycline cardiotoxicity 13 On the other hand, the release of inflammatory factors triggered by meningococcal disease, such as interferon gamma (IFN-γ), has also been reported to cause cardiac damage. 14 So is it the chemotherapy drugs themselves that cause cardiac energy metabolism disorders in mice? Or is it certain toxic cytokines released after chemotherapy destroys tumor cells that cause abnormal cardiac energy metabolism?

[0188] In order to exclude the influence of leukemia cells, the inventors first performed chemotherapy in normal mice without leukemia according to the induction chemotherapy regimen of the previous experiment, and observed the direct effects of chemotherapy drugs on the cardiac energy metabolism and cardiac function of mice. The inventors divided healthy tumor-free mice into two groups, one of which was treated with DNR chemotherapy and the other with PBS as a control, and then performed cardiac ultrasound and PET-CT tests ( Fig. 9 Interestingly, the inventors found that cardiac metabolism and function in healthy tumor-free mice did not change significantly after chemotherapy ( Fig. 9 BC part), the content of cardiac ATP did not show a significant decrease ( Fig. 9Therefore, the disorder of cardiac energy metabolism after chemotherapy may not be the direct effect of chemotherapy drugs, but the comprehensive result of the interaction between chemotherapy drugs and leukemia cells. Furthermore, the inventors conducted transcriptomic sequencing on the heart tissue of leukemia mice after chemotherapy, and the results showed that the cytokine and cytokine receptor interaction pathways were significantly enriched in the myocardial tissue after chemotherapy ( Fig.10 ), this result suggests that chemotherapy may cause some tumor-derived cytokines to be released into the blood, leading to energy metabolism disorders in the heart.

[0189] Based on the above assumptions, the inventors used Luminex multifactor detection technology to perform high-throughput screening of cytokines that changed in the plasma of leukemia mice and normal tumor-free mice after chemotherapy. The results showed that among all the cytokines detected, 9 cytokines increased significantly after chemotherapy in leukemia mice. Since the specific cytokines released by tumor cells need to conform to the change pattern of increasing after chemotherapy in leukemia mice but not significantly increasing after chemotherapy in healthy tumor-free mice, the inventors verified these 9 cytokines one by one, and the results showed that only interleukin 1α (IL-1α) met this trend characteristic ( Fig.11 ). The IL-1 family is divided into two subtypes: IL-1α and IL-1β. In the field of cardiovascular disease research, many studies have reported the effects of IL-1β on cardiomyocytes. 15 However, the research on IL-1α mainly focuses on its effects on cardiac vascular endothelial cells and fibroblasts, and there are few studies on its direct effects on cardiomyocytes. It is currently believed that IL-1α is a damage-related pattern molecule that is constitutively expressed in blood or non-blood cells. After cell necrosis, IL-1α is released into the intercellular space or blood circulation to exert its biological functions locally or systemically. 16 The inventors' previous in vitro experiments showed that DNR can induce leukemia cell necrosis, and the necrosis ratio increases in a dose-dependent manner ( Fig.12 In addition, the inventors also found that the protein expression of IL-1α in the culture supernatant of leukemia cells increased significantly after the addition of DNR ( Fig.12 Therefore, IL-1α released by leukemia cell necrosis induced by chemotherapy drugs may be a potential cause of cardiometabolic disorders and cardiac function damage.

[0190] In order to verify the role of IL-1α in cardiac metabolism and function, the inventors directly injected IL-1α recombinant protein into leukemia-free mice and found that IL-1α had a direct damaging effect on the heart ( Fig.13). The inventors further used cardiac-specific IL-1α receptor (IL1R1) knockout mice to construct a leukemia model and found that cardiac IL-1α receptor knockout protected cardiac metabolism and function in leukemia mice after chemotherapy. The above two experiments confirmed the key role of IL-1α in cardiac damage after chemotherapy from both positive and negative aspects ( Fig.14 ). At the same time, the inventors collected blood samples from children with leukemia after chemotherapy and evaluated their plasma IL-1α concentration and cardiac function before and after chemotherapy, proving that the increase in IL-1α in children was correlated with the decrease in cardiac function ( Fig.15 ).

[0191] In order to verify the source of IL-1α, the inventors used systemic IL-1α knockout mice to construct a leukemia model. At the same time, the inventors knocked down the IL-1α gene in leukemia primary cells using short hairpin RNA (shRNA) technology, and then used IL-1α knockdown cell lines to construct a leukemia model. Finally, by detecting the changes in plasma IL-1α content in the two models after chemotherapy, it was determined that the increase in plasma IL-1α after chemotherapy originated from the destruction and release of tumor cells ( Fig.16 ).

[0192] Example 3 NF-KappaB signaling pathway mediates IL-1α-induced cardiac injury

[0193] So how does the increase in plasma IL-1α levels after chemotherapy cause cardiometabolic disorders? Existing studies have shown that activation of IL-1α receptor IL1R1 can further activate the downstream NF-KappaB signaling pathway, thereby regulating gene expression. 17 . It is generally believed that NF-KappaB signaling is a common pathway for many inflammatory cytokines to exert their biological functions, and plays an important role in the progression of myocardial infarction and heart failure. The NF-KappaB protein family exists in the form of a heterodimer complex, among which p50 / p65 is the most important NF-KappaB complex in the heart. The p65 subunit is a transcription factor that can enter the cell nucleus after phosphorylation to regulate the transcription of downstream genes. 18 Recent studies have shown that p65 plays an important role in regulating a variety of metabolic genes in cells.

[0194] Transcriptomics results suggest that the cardiac NF-KappaB signaling pathway is significantly enriched in leukemia mice after chemotherapy ( Fig.10Further, the inventors preliminarily verified the activation level of the NF-KappaB signaling pathway in the hearts of leukemia mice after chemotherapy by immunoblotting. The results showed that the phosphorylation level of p65 in the heart increased and the expression level of the NF-KappaB inhibitory protein IkBα decreased. However, the activation level of the NF-KappaB signaling pathway in the hearts of healthy tumor-free mice after chemotherapy did not change significantly ( Fig.17 In addition, the inventors previously conducted immunofluorescence and immunoblotting tests on myocardial tissues of leukemia patients after chemotherapy, and the results showed that IL-1α was enriched in the myocardial tissue space after chemotherapy, accompanied by an increase in the activation level of the NF-KappaB signaling pathway ( Fig.18 ). Therefore, the inventors speculated that the increase in plasma IL-1α caused by chemotherapy may activate the NF-KappaB signaling pathway of cardiomyocytes, thereby affecting the expression of downstream metabolic genes.

[0195] To verify this hypothesis, the inventors used PDTC, an inhibitor of the NF-KappaB signaling pathway, to intervene in leukemia mice. Compared with the control group, the cardiac metabolism and function of the PDTC intervention group were significantly improved after chemotherapy ( Fig.19 ).

[0196] Example 4 PGC-1α reduction is the core of IL-1α-mediated cardiometabolic disorders

[0197] PGC-1α is a transcriptional coactivator that can bind to a variety of transcription factors and plays an important role in fatty acid oxidation, glycolysis, mitochondrial biogenesis, etc. 19 In recent years, PGC-1α has been found to be closely related to the energy metabolism of the heart. Studies have shown that decreased expression of PGC-1α will inhibit fatty acid metabolism in the heart and further induce abnormal cardiac function. 20 Studies have shown that activation of the NF-KappaB signaling pathway can downregulate the expression of PGC-1α in cells, ultimately changing the cell metabolic pattern. 21 .

[0198] In previous studies, the inventors used the Ingenuity Pathway Analysis system to enrich the cardiac transcriptomics data of leukemia mice after chemotherapy. The results showed that the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which is closely related to metabolism, was significantly downregulated ( Fig.10 Part B). Further, the inventors conducted a protein interaction network analysis on the differentially expressed genes in the PPAR pathway and found that PGC-1α and p65 interacted with each other among the central genes ( Fig.10Then, the inventors detected the expression changes of PGC-1α in the heart of leukemia mice after chemotherapy at the transcriptional and translational levels by RT-PCR and immunoblotting. The results showed that the expression of PGC-1α in the heart of leukemia mice after chemotherapy was significantly downregulated ( Fig. 20 ). In addition, the inventors' previous single-cell transcriptomics results showed that the expression level of PGC-1α in cardiomyocytes of leukemia children decreased after chemotherapy, and its expression level determined the different fates of cardiomyocytes after chemotherapy ( Fig.21 ). The inventors therefore speculated that the activation of the myocardial NF-KappaB signaling pathway after chemotherapy may cause cardiac energy metabolism disorders and functional damage by inhibiting the expression of PGC-1α.

[0199] The inventors demonstrated through dual luciferase reporter experiments that p65 can bind to the promoter region of the PGC-1α gene and inhibit the transcription of PGC-1α ( Fig. 22 ). Using adeno-associated virus type 9 (AAV9-PGC-1α) carrying a myocardial-specific promoter and PGC-1α gene coding sequence to specifically overexpress PGC-1α in the hearts of leukemia mice, the cardiac energy metabolism disorder and functional abnormality after chemotherapy were corrected, thereby proving the core role of PGC-1α in chemotherapy-related cardiac dysfunction ( Fig.23 ).

[0200] Example 5 IL-1α neutralizing antibody alleviates cardiac damage and metabolic disorders caused by leukemia treatment

[0201] After clarifying the mechanism of cardiac damage caused by leukemia treatment (chemotherapy or immunotherapy, etc.), the inventors believe that IL-1α neutralizing antibodies have translational value and broad application prospects in improving cardiac damage in patients. IL-1α plays an important role in both leukemia occurrence and cardiac damage. Blocking the IL-1α-mediated cardiac NF-KappaB / PGC-1α axis may reduce the potential risk of cardiac toxicity.

[0202] The present invention found that the use of 10 μg / mouse IL-1α neutralizing antibody (Mouse IL-1alpha / IL-1F1antibody, catalog number: AB-400-NA, supplier: R&D Systems, product website: https: / / www.rndsystems.com / cn / products / mouse-il-1alpha-il-1f1-antibody_ab-400-na#product-details) can reduce cardiac metabolic and functional damage in leukemia mice (AML, T-ALL or B-ALL) after treatment without affecting the therapeutic effect ( Fig.24 , Fig.26 and Fig. 27 ).

[0203] Example 6 IL1R1 antagonists alleviate cardiac damage and metabolic disorders caused by leukemia treatment

[0204] The present invention found that the use of IL1R1 antagonist (Anakinra, CAS No.: 143090-92-0) at a dose of 30 mg / kg / d×5d can reduce the cardiac metabolism and functional damage of leukemia mice (AML mice) after treatment without affecting the therapeutic effect ( Fig.28 ).

[0205] summary

[0206] The introduction of the concept of "oncology cardiology" has made the complications of heart damage caused by anti-tumor treatment more and more concerned. However, many studies currently focus on the heart damage directly caused by chemotherapy drugs, but ignore the interaction between tumors and chemotherapy drugs, and the difference in tumor load levels that may cause heart damage after chemotherapy. The present invention found that there is a close relationship between tumor cells, chemotherapy drugs, and heart damage. Chemotherapy releases IL-1α by inducing tumor cell necrosis, which acts on the myocardial IL1R1 receptor and activates the NF-KappaB signaling pathway, thereby inhibiting the expression of PGC-1α and ultimately leading to cardiac energy metabolism disorders and abnormal cardiac function ( Fig.25 ).

[0207] The number of childhood leukemia survivors increases year by year, and life expectancy continues to increase. The long-term cardiovascular burden continues to increase, and cardiovascular health problems caused by chemotherapy seriously affect their quality of life. At present, there is still a lack of effective protection strategies for leukemia chemotherapy-related cardiac damage. Early identification of cardiac pathological and physiological changes caused by leukemia chemotherapy and active intervention will effectively reduce the risk of cardiovascular system diseases in children with leukemia after chemotherapy. It is urgent to develop individualized peri-chemotherapy cardiovascular protection strategies based on the unique spectrum of tumor diseases and physiological characteristics of children. This will effectively reduce cardiovascular complications during chemotherapy, reduce the incidence of long-term adverse cardiovascular events, and benefit many children with leukemia, thereby achieving significant social and economic effects.

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Claims

1. Application of IL-1α inhibitors in the preparation of drugs for treating cardiac damage induced by leukemia treatment.

2. The use according to claim 1, characterized in that The leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; Preferably, the leukemia treatment satisfies one or more of the following conditions: The chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; The leukemia treatment results in necrosis of leukemia cells; and, The immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy.

3. The use according to claim 1, characterized in that The leukemia is acute myeloid leukemia or acute lymphocytic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; and / or, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure; Preferably, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and / or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

4. The use according to claim 1, characterized in that The IL-1α inhibitor is an anti-IL-1α antibody; Preferably, the anti-IL-1α antibody is a monoclonal antibody and / or a neutralizing antibody; More preferably, the anti-IL-1α antibody is purchased from R&D Systems with the product number AB-400-NA.

5. Use of an antibody purchased from R&D Systems with catalog number AB-400-NA in the preparation of a drug for treating cardiac damage induced by leukemia treatment; Preferably, the leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; and / or, the leukemia is acute myeloid leukemia or acute lymphocytic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; More preferably, the leukemia treatment causes leukemia cell necrosis; and / or, the immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy.

6. The use according to claim 5, characterized in that The chemotherapy drug is an anthracycline chemotherapy drug, preferably daunorubicin; and / or, the acute myeloid leukemia is caused by MLL-AF9 fusion protein.

7. The use according to claim 5, characterized in that The cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure; Preferably, the cardiometabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

8. Use of IL1R1 antagonists in the preparation of drugs for treating cardiac damage induced by leukemia treatment; Preferably, the antagonist of IL1R1 is a competitive inhibitory drug or a blocking drug of IL1R1; More preferably, the competitive inhibitory drug is Anakinra.

9. The use according to claim 8, characterized in that The leukemia treatment method is selected from: one or more of chemotherapy, radiotherapy, molecular targeted therapy and immunotherapy; Preferably, the leukemia treatment satisfies one or more of the following conditions: The chemotherapy drug is an anthracycline chemotherapy drug, such as daunorubicin; The leukemia treatment results in necrosis of leukemia cells; and, The immunotherapy is CAR-T therapy, such as CD19 CAR-T therapy.

10. The use according to claim 8, characterized in that The leukemia is acute myeloid leukemia or acute lymphocytic leukemia, such as acute lymphoblastic T-cell leukemia or acute lymphoblastic B-cell leukemia; and / or, the cardiac injury is selected from one or more of: cardiac metabolic disorder, abnormal cardiac function, cardiomyopathy and heart failure; Preferably, the acute myeloid leukemia is caused by MLL-AF9 fusion protein; and / or the cardiac metabolic disorder is decreased myocardial fatty acid metabolism and enhanced glucose metabolism.

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