Application of FSP1 as prognostic marker or therapeutic target of non-early precursor T acute lymphocytic leukemia

Abstract

The invention provides application of FSP1 as a prognostic marker or a therapeutic target of non-early precursor T acute lymphocytic leukemia (non-ETP ALL), and belongs to the technical field of biological medicine. The invention discovers that the high expression of the coding gene AIFM2 of the FSP1 protein prompts that the non-ETP ALL patient is poor in prognosis. The FSP1 is used as a therapeutic target, and the FSP1 inhibitor not only can remarkably kill non-ETP ALL cells, but also can synergistically increase the sensitivity of the non-ETP ALL cells to small-dose cytarabine (Ara-C). In addition, the FSP1 inhibitor is combined with a small dose of Ara-C, so that the infiltration proportion of non-ETP ALL cells in the spleen and bone marrow of the mouse can be obviously reduced. The AIFM2 gene and the FSP1 protein coded by the AIFM2 gene are expected to become a prognostic marker and a therapeutic target of non-ETP ALL.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of FSP1 as a prognostic marker or therapeutic target for non-early precursor T-cell acute lymphoblastic leukemia. Background Technology

[0002] Leukemia is a type of malignant clonal disease of hematopoietic stem cells. Due to mechanisms such as differentiation disorders, uncontrolled proliferation, and inhibited apoptosis, malignant cells proliferate in large numbers in the bone marrow and other hematopoietic tissues, leading to severe clinical symptoms and complications. Among them, acute T-lymphoblastic leukemia (T-ALL) is a highly malignant subtype of leukemia, accounting for approximately 15% and 25% of acute lymphoblastic leukemia cases in children and adults, respectively. Due to its high heterogeneity, tendency to invade the central nervous system, insensitivity to conventional chemotherapy, and high relapse rate, T-ALL is clinically classified as a subtype with a poor prognosis. In terms of risk stratification, T-ALL is divided into early T-cell precursor ALL (ETP-ALL) and non-ETP ALL based on different immunophenotypes, with the former being a high-risk subtype with an extremely poor prognosis. Although the prognosis of non-ETP ALL patients is relatively better than that of ETP-ALL patients, some patients still experience drug resistance and relapse, and there is a lack of widely accepted molecular prognostic biomarkers and therapeutic targets for non-ETP ALL patients in clinical practice. Summary of the Invention

[0003] The purpose of this invention is to provide the application of FSP1 as a prognostic biomarker or therapeutic target for non-early precursor T-cell acute lymphoblastic leukemia, and to provide new ideas and strategies for the prognostic assessment and clinical treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0004] This invention provides a reagent or method for detecting the expression level of FSP1 protein or the gene encoding FSP1 protein in the preparation of a product for prognostic assessment of non-early precursor T-cell acute lymphoblastic leukemia; the amino acid sequence of the FSP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the FSP1 protein is shown in SEQ ID NO.2.

[0005] Preferably, the reagent for detecting the expression level of FSP1 protein includes an antibody that specifically binds to FSP1; the method for detecting the expression level of FSP1 protein includes Western blotting.

[0006] Preferably, the prognostic assessment metrics include overall survival and / or event-free survival.

[0007] The present invention also provides the use of FSP1 inhibitors in the preparation of drugs for treating non-early precursor T-cell acute lymphoblastic leukemia.

[0008] The present invention also provides the use of FSP1 inhibitors in the preparation of drugs that improve the sensitivity of cytarabine to the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0009] The present invention also provides the application of FSP1 inhibitors in combination with cytarabine in the preparation of drugs for the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0010] Preferably, the FSP1 inhibitor comprises at least one of the following: 1) a substance that inhibits the activity of FSP1 protein; 2) a substance that reduces the content of FSP1 protein; 3) silencing, knocking out, or mutating. AIFM2 Genetic material; 4) Interference or inhibition AIFM2 Substances involved in gene expression.

[0011] Preferably, the substance that inhibits the activity of FSP1 protein includes an FSP1 antibody, a peptide, or a small molecule compound; The substances that reduce FSP1 protein content include substances that promote FSP1 protein degradation.

[0012] Preferably, the dosage form of the drug is an injection.

[0013] The present invention also provides a medicament for treating non-early precursor T-cell acute lymphoblastic leukemia, the active ingredients of which include FSP1 inhibitor and cytarabine.

[0014] This invention provides a reagent or method for detecting the expression level of FSP1 protein or its encoding gene in the preparation of products for prognostic assessment of non-early precursor T-cell acute lymphoblastic leukemia; the amino acid sequence of the FSP1 protein is shown in SEQ ID NO.1; the nucleotide sequence of the encoding gene of the FSP1 protein is shown in SEQ ID NO.2. This invention discovers the encoding gene of the FSP1 protein. AIFM2 High expression suggests a poor prognosis for patients with non-ETP ALL. Targeting FSP1, FSP1 inhibitors not only significantly kill non-ETP ALL cells but also synergistically increase their sensitivity to low-dose cytarabine (Ara-C). Furthermore, the combination of FSP1 inhibitors and low-dose Ara-C significantly reduced the infiltration rate of non-ETP ALL cells in the spleen and bone marrow of mice. AIFM2 The gene and its encoded FSP1 protein are expected to become a prognostic biomarker and therapeutic target for non-ETPALL. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Results of Kaplan-Meier analysis of overall survival curves in non-ETP ALL patients; Figure 2 Results of Kaplan-Meier analysis of event-free survival curves in non-ETP ALL patients; Figure 3 To adopt AIFM2 The results of receiver operating characteristic (ROC) curves for assessing gene expression levels in non-ETP ALL patients with 10-year overall survival; Figure 4 To adopt AIFM2 The results of receiver operating characteristic (ROC) curves for assessing gene expression levels in non-ETP ALL patients with 10-year event-free survival; Figure 5 for AIFM2 Gene expression levels in different risk groups; Figure 6 The survival rate and IC50 of non-ETP ALL cells treated with different concentrations of FSP1 inhibitors are statistically shown. The experimental data are expressed as mean ± standard deviation. The FSP1 inhibitor treatment groups at different concentrations are compared with the corresponding DMSO treatment control groups. The values ​​in the table on the right are the IC50 values ​​of CCRF-CEM (top) and MOLT-4 (bottom) cells treated with FSP1 inhibitors, respectively. Figure 7 The survival rate and IC50 of non-ETP ALL cells treated with different concentrations of Ara-C combined with FSP1 inhibitors are statistically analyzed. The experimental data are expressed as mean ± standard deviation. The Ara-C combined with FSP1 inhibitor treatment groups were compared with the corresponding Ara-C monotherapy treatment groups. The values ​​on the left side of the table on the right are the IC50 values ​​of CCRF-CEM (top) and MOLT-4 (bottom) cells treated with Ara-C monotherapy, respectively; the values ​​on the right side are the IC50 values ​​of CCRF-CEM (top) and MOLT-4 (bottom) cells treated with Ara-C combined with FSP1 inhibitors, respectively. Figure 8The graph shows the combined index of Ara-C and FSP1 inhibitor drugs and the calculation results; in the graph above, the horizontal axis Fa represents the inhibition rate and the vertical axis CI represents the combined index; the table below lists the CI values ​​of 0.2 nM, 0.5 nM and 1 nM Ara-C combined with 7.5 μM FSP1 inhibitor for CCRF-CEM (top) and MOLT-4 (bottom) cells respectively. Figure 9 A flowchart of the dosing strategy for treating non-ETP ALL mouse models with Ara-C monotherapy or in combination with FSP1 inhibitors; Figure 10 Images showing spleen size in non-ETP ALL mice from different drug treatment groups, with a reference scale at the bottom of the images; Figure 11 Representative flow cytometry images of leukemia cell infiltration in the spleen (top) and bone marrow (bottom) of non-ETP ALL mice in different drug treatment groups; GFP detected by flow cytometry. + The percentage accurately reflects the proportion of leukemia cells infiltrating the spleen and bone marrow of mice; the cells within the box in the figure are GFP. + Cell population, the numbers shown are GFP + percentage; Figure 12 Statistical results of leukemia cell infiltration in the spleen (left) and bone marrow (right) of non-ETP ALL mice in different drug treatment groups; experimental data are taken as mean ± standard error. Among them, the Ara-C combined with FSP1 inhibitor treatment group was compared with the saline or DMSO control group and the Ara-C single drug treatment group. Detailed Implementation

[0017] This invention provides the application of reagents or methods for detecting the expression level of FSP1 protein or the gene encoding FSP1 protein in the preparation of products for prognostic assessment of non-early precursor T-cell acute lymphoblastic leukemia; The amino acid sequence of the FSP1 protein is shown in SEQ ID NO.1, specifically as follows: MGSQVSVESGALHVVIVGGGFGGIAAASQLQALNVPFMLVDMKDSFHHNVAALRASVETGFAKKTFISYSVTFKDNFRQGLVVGIDLKNQMVLLQGGEALPFSHLILATGSTGPFPGKFNEVSSQQAAIQAYEDMVRQVQRSRFIVVVGGGSAGVEMAAEIKTEYPEKEVTLIHSQVALADKELLPS VRQEVKEILLRKGVQLLLSERVSNLEELPLNEYREYIKVQTDKGTEVATNLVILCTGIKINSSAYRKAFESRLASSGALRVNEHLQVEGHSNVYAIGDCADVRTPKMAYLAGLHANIAVANIVNSVKQRPLQAYKPGALTFLLSMGRNDGVGQISGFYVGRLMVRLTKSRDLFVSTSWKTMRQSPP; The nucleotide sequence of the gene encoding the FSP1 protein is shown in SEQ ID NO.2, specifically as follows:

[0018] This invention first explored AIFM2 The impact of gene expression levels on the prognosis of non-ETP ALL patients. Step 1: Transcriptome sequencing data and clinical information of 863 non-ETP ALL patients were downloaded from the Synapse database (accession number: syn54032669, https: / / doi.org / 10.7303 / syn54032669). Step 2: Input... AIFM2 Gene expression levels and overall survival data were used. The optimal cutoff point for overall survival, 6.4896, was calculated using the `surv_cutpoint` function of the `survminer` package. Based on this cutoff point, non-ETP ALL patients were divided into a high-expression group (n=711, expression level 6.4921~8.5797) and a low-expression group (n=152, expression level 5.2665~6.4896). Similarly, input... AIFM2 Gene expression levels and event-free survival data were analyzed. The optimal cutoff point for event-free survival, 6.5036, was calculated using the `surv_cutpoint` function of the `survminer` package. Based on this cutoff point, non-ETP ALL patients were divided into a high-expression group (n=700, expression level 6.5037~8.5797) and a low-expression group (n=163, expression level 5.2665~6.5036). Thirdly, Kaplan-Meier survival curves were used to plot the overall survival and event-free survival curves for both groups, and log-rank and hazard ratio (HR) analyses were performed. Results showed that compared to... AIFM2 Low gene expression group AIFM2 In the gene-overexpression group, non-ETP ALL patients had significantly reduced overall survival and event-free survival, and a significantly increased risk of death. Fourth step, based on... AIFM2 Non-ETP ALL patients were divided into a high-expression group (n=259, expression range: 7.116~8.5797) and a low-expression group (n=259, expression range: 5.2665~6.6493) using 30% above and below the gene expression level as cutoff points. Receiver operating characteristic (ROC) curves were plotted for both groups, and the area under the curve (AUC) for 10-year overall survival and event-free survival was calculated. Results showed that... AIFM2 The area under the curve (AUC) for assessing 10-year overall survival in non-ETP ALL patients based on gene expression levels was 0.695. AIFM2 The area under the curve (AUC) for assessing 10-year event-free survival in non-ETP ALL patients based on gene expression levels was 0.683, indicating good diagnostic value. The fifth step involved grouping non-ETP ALL patients according to their risk profile and comparing... AIFM2 Gene expression levels in different risk groups. Results showed that compared to the low-risk group (n=86), AIFM2 Gene expression levels were significantly increased in the high-risk group and patients who failed induction therapy (n=120); similarly, compared with patients in the intermediate-risk group (n=645), AIFM2 Gene expression levels were also significantly increased in high-risk patients and patients who failed induction therapy (n=120).

[0019] As one implementation, the reagent for detecting the expression level of FSP1 protein includes an antibody that specifically binds to FSP1, further comprising an FSP1 monoclonal antibody purchased from Cell Signaling Technology, Inc., USA, catalog number: 51676T; the method for detecting the expression level of FSP1 protein includes Western blotting.

[0020] As one implementation, the prognostic assessment metrics include overall survival and / or event-free survival.

[0021] In one implementation, the product includes reagents or kits.

[0022] In one implementation method, the present invention uses 6.5036 as a threshold. When the expression level of FSP1 protein is greater than or equal to 6.5036, it indicates a poor prognosis, and vice versa.

[0023] The present invention also provides the use of FSP1 inhibitors in the preparation of drugs for treating non-early precursor T-cell acute lymphoblastic leukemia.

[0024] As one implementation, when the FSP1 inhibitor is used alone to treat non-early precursor T acute lymphoblastic leukemia cell lines in vitro, the working concentration of the FSP1 inhibitor is 5-50 μM, further 10-30 μM, and even further 20 μM.

[0025] In one embodiment, the non-early precursor T-cell acute lymphoblastic leukemia cell line is either the human non-ETPALL cell line CCRF-CEM or the human non-ETPALL cell line MOLT-4.

[0026] by AIFM2 The FSP1 protein encoded by the gene is the therapeutic target. This invention detected different concentrations of FSP1 inhibitors (C... 20 H 13The killing effects of FSP1 inhibitors (5 μM, 10 μM, 20 μM, 30 μM, and 50 μM) on non-ETP ALL cell lines. Results showed that in the human non-ETP ALL cell line CCRF-CEM, treatment with 5 μM, 10 μM, 20 μM, 30 μM, and 50 μM FSP1 inhibitors resulted in mean cell viability of 89.35%, 78.13%, 63.39%, 49.72%, and 25.07%, respectively. In another human non-ETP ALL cell line, MOLT-4, treatment with 5 μM, 10 μM, 20 μM, 30 μM, and 50 μM FSP1 inhibitors resulted in mean cell viability of 72.9%, 65.57%, 54.16%, 37.3%, and 17.43%, respectively. The median inhibition concentration (IC50) of the FSP1 inhibitor was further calculated using Graphpad software. The results showed that the IC50 of the FSP1 inhibitor in the CCRF-CEM and MOLT-4 cell lines were 27.11 μM and 17.54 μM, respectively.

[0027] The present invention also provides the use of FSP1 inhibitors in the preparation of drugs that improve the sensitivity of cytarabine to the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0028] As one implementation, the improvement of the sensitivity of cytarabine to non-early precursor T-cell acute lymphoblastic leukemia includes improving the sensitivity of low-dose cytarabine to non-early precursor T-cell acute lymphoblastic leukemia; the dosage of cytarabine used in mice is 30 mg / kg.

[0029] The present invention also provides the application of FSP1 inhibitors in combination with cytarabine in the preparation of drugs for the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0030] As one implementation, the FSP1 inhibitor includes at least one of the following: 1) Substances that inhibit the activity of FSP1 protein; 2) Substances that reduce FSP1 protein levels; 3) Silencing, knockout, or mutation AIFM2 Genetic material; 4) Interference or suppression AIFM2 Substances involved in gene expression.

[0031] As one implementation method, the substance inhibiting FSP1 protein activity is an FSP1 selective inhibitor with the chemical formula C. 20 H 13N5, with the chemical structure shown in Formula 1, CAS#150651-39-1; its inhibition mechanism is to competitively bind to FSP1 with NADH to inhibit its enzyme activity.

[0032] Formula 1.

[0033] In one embodiment, the substance that inhibits the activity of FSP1 protein includes FSP1 antibody, peptide, or small molecule compound; the substance that reduces the content of FSP1 protein includes substances that promote the degradation of FSP1 protein.

[0034] In one embodiment, when the FSP1 inhibitor is combined with cytarabine for in vitro treatment of non-early precursor T acute lymphoblastic leukemia cell lines, the working concentration of the FSP1 inhibitor is 7.5 μM; the working concentration of the cytarabine is 0.2~1 nM, more specifically 0.5 nM.

[0035] This invention further investigated the combined efficacy of an FSP1 inhibitor (7.5 μM) with low-dose Ara-C (0.2 nM, 0.5 nM, and 1 nM) in non-ETP ALL cell lines. The results showed that in CCRF-CEM cells, treatment with 0.2 nM, 0.5 nM, and 1 nM Ara-C alone resulted in mean leukemia cell survival rates of 76.48%, 60.04%, and 36.78%, respectively. Targeting FSP1, treatment of CCRF-CEM cells with a 7.5 μM FSP1 inhibitor in combination with 0.2 nM, 0.5 nM, and 1 nM Ara-C resulted in mean leukemia cell survival rates of 49.31%, 42.97%, and 36.89%, respectively. In MOLT-4 cells, the mean survival rates of leukemia cells treated with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 91.34%, 66.70%, and 38.21%, respectively. Targeting FSP1, the mean survival rates of leukemia cells treated with 7.5 μM FSP1 inhibitor in combination with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 55.71%, 45.79%, and 33.11%, respectively. Further calculation of the IC50 of Ara-C using Graphpad software showed that the IC50 values ​​of Ara-C monotherapy for CCRF-CEM and MOLT-4 cells were 833.9 nM and 976.2 nM, respectively; when combined with an FSP1 inhibitor, the IC50 values ​​of Ara-C decreased to 213 nM and 285.8 nM, respectively. Finally, the combination index (CI) was calculated using Compusyn software to assess the combined effect of the FSP1 inhibitor and Ara-C. The results showed that in CCRF-CEM cells, the CI values ​​for 7.5 μM FSP1 inhibitor combined with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 0.5069, 0.5703, and 0.6311, respectively, all less than 1; in MOLT-4 cells, the CI values ​​for 7.5 μM FSP1 inhibitor combined with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 0.4429, 0.5421, and 0.6689, respectively, all less than 1.

[0036] As one implementation method, when the FSP1 inhibitor is used in combination with cytarabine to treat non-early precursor T-cell acute lymphoblastic leukemia, the dosage of the FSP1 inhibitor is 5 mg / kg and the dosage of the cytarabine is 30 mg / kg in mice.

[0037] Finally, this invention tested the antileukemic efficacy of the FSP1 inhibitor (5 mg / kg) and a low dose of Ara-C (30 mg / kg) in a non-ETPALL mouse model. First, mice were injected intravenously with GFP-positive (GFP-positive) mice. + CCRF-CEM cells (2×10⁻⁶) 6 A non-ETP ALL mouse model was constructed using cells per mouse. Starting on day 11, mice were intraperitoneally injected with an FSP1 inhibitor and / or a low dose of Ara-C (with saline or DMSO as negative controls) every other day for a total of 7 times. On day 4 after the injection, mice were sacrificed and their spleens and bone marrow were isolated to observe spleen size and organ infiltration. Results showed that compared with the saline or DMSO control group (n=7), the spleens of mice in the Ara-C monotherapy group (n=6), the FSP1 inhibitor monotherapy group (n=6), and the Ara-C + FSP1 inhibitor combination therapy group (n=6) were significantly smaller. Subsequently, flow cytometry was used to detect the infiltration of non-ETP ALL cells in the mouse spleen and bone marrow. The results showed that in the spleen of mice, the mean percentage of leukemia cell infiltration was 7.73% in the saline or DMSO control group; 5.91% in the Ara-C monotherapy group; and 4.02% in the Ara-C + FSP1 inhibitor combination therapy group. In the bone marrow of mice, the mean percentage of leukemia cell infiltration was 10.62% in the saline or DMSO control group; 1.66% in the Ara-C monotherapy group; and only 0.42% in the Ara-C + FSP1 inhibitor combination therapy group.

[0038] In one embodiment, the dosage form of the drug is an injection, further including an intraperitoneal injection.

[0039] As one implementation, the treatment of non-early precursor T-cell acute lymphoblastic leukemia includes reducing the infiltration of leukemia cells in the bone marrow and / or spleen of patients with non-early precursor T-cell acute lymphoblastic leukemia.

[0040] The present invention also provides a medicament for treating non-early precursor T-cell acute lymphoblastic leukemia, the active ingredients of which include FSP1 inhibitor and cytarabine.

[0041] In one embodiment, the active ingredients of the drug are an FSP1 inhibitor and cytarabine; the mass ratio of the FSP1 inhibitor to cytarabine is 1:6. In this invention, there is a synergistic effect between the FSP1 inhibitor and Ara-C.

[0042] In one embodiment, the drug includes an injectable form, and more particularly, an intraperitoneal injectable form.

[0043] This invention proves AIFM2 High gene expression suggests a poor prognosis in non-ETP ALL patients; AIFM2 The FSP1 protein encoded by the gene serves as a therapeutic target. FSP1 inhibitors not only significantly kill non-ETP ALL cells but also synergistically increase the sensitivity of non-ETP ALL cells to low-dose Ara-C. Furthermore, the combination of FSP1 inhibitors and low-dose Ara-C significantly reduces the proportion of leukemia cell infiltration in the spleen and bone marrow of mice. This invention provides a novel prognostic biomarker and therapeutic target for non-ETP ALL, with broad application prospects in the field of medical research.

[0044] To further illustrate the present invention, the application of FSP1 provided by the present invention as a prognostic marker or therapeutic target for non-early precursor T-cell acute lymphoblastic leukemia is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0045] All experiments in the following examples were performed in triplicate. Results for cell line experiments were expressed as mean ± standard deviation; results for mouse experiments were expressed as mean ± standard error. Statistical analysis was performed using t-tests. This indicates that the p-value is less than 0.05; This indicates that the p-value is less than 0.01; This indicates that the p-value is less than 0.001.

[0046] The human non-ETP ALL cell lines CCRF-CEM and MOLT-4 in the following examples were purchased from Beina Biotechnology Co., Ltd. and Kebai Biotechnology Co., Ltd., respectively. The origin of both cell lines was confirmed by cellular STR identification, and they were confirmed to be free of mycoplasma contamination.

[0047] The experimental animals used in the following examples were nonobese diabetic / severe combined immune deficiency (NON / SCID) mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0048] The chemotherapy drug cytarabine (Ara-C) in the following examples is cytarabine for injection manufactured by Pfizer Inc., batch number HL1419. The FSP1 inhibitor is a product of MedChemExpress, catalog number HY-136057, with the chemical formula C. 20 H 13 N5, CAS#150651-39-1.

[0049] The RPMI-1640 medium used in the following examples is a Gibco product, and the fetal bovine serum (FBS) is a Sigma product.

[0050] In the following examples, the half-maximal inhibitory concentration (IC50) refers to the drug concentration at which 50% of tumor cell growth is inhibited.

[0051] In the following examples, the drug combination index (CI) refers to the degree of drug interaction quantitatively measured at a certain effect endpoint during combination chemotherapy. If CI is less than 1, it indicates a synergistic effect between the drugs; if CI equals 1, it indicates an additive effect between the drugs; if CI is greater than 1, it indicates an antagonistic effect between the drugs.

[0052] Example 1 Overall survival and event-free survival in non-ETP ALL patients 1. Download transcriptome sequencing data and clinical data of 863 non-ETP ALL patients from the Synapse database (accession number: syn54032669, https: / / doi.org / 10.7303 / syn54032669); input... AIFM2 Gene expression levels and overall survival data were used to calculate the optimal cutoff point for overall survival (6.4896) using the surv_cutpoint function of the survminer package. Based on this cutoff point, non-ETP ALL patients were divided into... AIFM2 The gene-overexpressing group (n=711, expression levels 6.4921~8.5797) and AIFM2 The gene expression low group (n=152, expression level 5.2665~6.4896); similarly, input... AIFM2 Gene expression levels and event-free survival data were used to calculate the optimal event-free survival cutoff point of 6.5036 using the surv_cutpoint function of the survminer package. Based on this cutoff point, non-ETP ALL patients were divided into... AIFM2 The gene-overexpressing group (n=700) had expression levels ranging from 6.5037 to 8.5797. AIFM2 The gene expression low group (n=163, expression level 5.2665~6.5036) was included. Kaplan-Meier survival curves were used to plot the overall survival curves and event-free survival curves for the two groups of patients, and log-rank test and hazard ratio (HR) analysis were performed.

[0053] The results are as follows Figure 1 and Figure 2 As shown. The results indicate that: compared to AIFM2 Low gene expression group AIFM2In the gene-overexpression group, non-ETP ALL patients had significantly reduced overall survival and event-free survival. Specifically, in the overall survival analysis... AIFM2 The risk of death in the gene-overexpression group was 1.85 times that of the control group; in the event-free survival analysis, AIFM2 The risk of death for patients in the gene-overexpression group was 1.88 times that of the control group.

[0054] 2. Take the result obtained in step 1 AIFM2 Non-ETP ALL patients were divided into a high-expression group (n=259, expression range: 7.116~8.5797) and a low-expression group (n=259, expression range: 5.2665~6.6493) using 30% above and below the gene expression level as cutoff points. ROC curves were plotted for both groups, and the results are as follows: Figure 3 and Figure 4 The results indicate that: AIFM2 The area under the curve (AUC) for assessing 10-year overall survival in non-ETP ALL patients using gene expression levels was 0.695, with a sensitivity of 88.2% and a specificity of 55.6%. AIFM2 The area under the curve (AUC) for assessing 10-year event-free survival in non-ETP ALL patients using gene expression levels was 0.683, with a sensitivity of 71.3% and a specificity of 66.7%. The above-mentioned overall survival and event-free survival... AIFM2 The gene expression threshold was 6.5036, i.e. AIFM2 A gene expression level greater than or equal to 6.5036 indicates a poor prognosis, while a level lower than 6.5036 indicates a good prognosis.

[0055] 3. Based on the results obtained in step 1 AIFM2 Further comparison of gene expression levels and clinical data. AIFM2 Gene expression levels in different risk groups, results are as follows: Figure 5 The results indicated that compared to the low-risk group (n=86), AIFM2 Gene expression levels were significantly increased in the high-risk group and patients who failed induction therapy (n=120); similarly, compared with patients in the intermediate-risk group (n=645), AIFM2 Gene expression levels were also significantly increased in high-risk patients and patients who failed induction therapy (n=120).

[0056] As can be seen from the above, AIFM2 Genes can serve as targets for prognostic assessment and clinical treatment of non-early precursor T-cell acute lymphoblastic leukemia.

[0057] Example 2 Detection of non-ETP ALL cell viability after chemotherapy treatment 1. Human non-ETP ALL cell lines CCRF-CEM and MOLT-4 were cultured in 1 mL of RPMI-1640 medium containing 10% FBS in 24-well plates at a cell density of 5 × 10⁶ cells / well. 4 Cells were added to each well with different concentrations of FSP1 inhibitor (5 μM, 10 μM, 20 μM, 30 μM and 50 μM). Cells were harvested after 24 h, and cell viability was detected using the CellTiter-Blue cell viability assay kit from Promega, USA. The IC50 of the drug was calculated using Graphpad software.

[0058] The results are as follows Figure 6 As shown in the figure. The results showed that in the human non-ETP ALL cell line CCRF-CEM, the mean survival rates of leukemia cells after treatment with 5 μM, 10 μM, 20 μM, 30 μM, and 50 μM FSP1 inhibitors were 89.35%, 78.13%, 63.39%, 49.72%, and 25.07%, respectively, with an IC50 of 27.11 μM for the FSP1 inhibitor. In another human non-ETP ALL cell line, MOLT-4, the mean survival rates of leukemia cells after treatment with 5 μM, 10 μM, 20 μM, 30 μM, and 50 μM FSP1 inhibitors were 72.9%, 65.57%, 54.16%, 37.3%, and 17.43%, respectively, with an IC50 of 17.54 μM for the FSP1 inhibitor.

[0059] 2. Human non-ETP ALL cell lines CCRF-CEM and MOLT-4 were cultured in 1 mL of RPMI-1640 medium containing 10% FBS in 24-well plates at a cell density of 5 × 10⁶ cells / well. 4 Cells / well, each type of test cell was divided into the following treatment groups according to the different drugs added: Treatment group 1: Different concentrations of Ara-C (0, 0.2 nM, 0.5 nM, 1 nM and 5 nM) were added respectively. Treatment group 2: Different concentrations of Ara-C (0, 0.2 nM, 0.5 nM, 1 nM and 5 nM) were added in combination with 7.5 μM FSP1 inhibitor; Cells were harvested 24 h after treatment. Cell viability was detected using the CellTiter-Blue cell viability assay kit from Promega, USA, and the IC50 of the drug was calculated using Graphpad software.

[0060] The results are as follows Figure 7As shown in the figure. The results showed that after treatment with different concentrations of Ara-C, the mean survival rates of CCRF-CEM cells were 76.48%, 60.04%, and 36.78%, respectively, and the IC50 of Ara-C was 833.9 nM. When combined with 7.5 μM FSP1 inhibitor, the mean survival rates of CCRF-CEM cells were 49.31%, 42.97%, and 36.89%, respectively, and the IC50 of Ara-C was 213 nM, a decrease of approximately 2.9-fold. Similarly, after treatment with different concentrations of Ara-C, the mean survival rates of MOLT-4 cells were 91.34%, 66.70%, and 38.21%, respectively, with an IC50 of 976.2 nM for Ara-C. When combined with 7.5 μM of FSP1 inhibitor, the mean survival rates of MOLT-4 cells were 55.71%, 45.79%, and 33.11%, respectively, with an IC50 of 285.8 nM for Ara-C, a decrease of approximately 2.4-fold. These results indicate that the combination with an FSP1 inhibitor can significantly increase the sensitivity of non-ETP ALL cells to low-dose Ara-C.

[0061] Example 3 Drug combination index detection of leukemia cells treated with combined chemotherapy drugs The drug concentrations of Ara-C and FSP1 inhibitors from step two of Example 2, along with the obtained cell viability data, were input into Compusyn software to calculate the drug combination index CI. The calculation formula is as follows: in, n Represents the number of drugs in a combination; (D x ) i represent i Drug inhibition alone x % of the dose; (D) i represent i Drugs inhibited in combination therapy x The percentage of the drug's concentration (CI) indicates the drug's synergistic effect. A CI less than 1 indicates a synergistic effect between the drugs; a CI equal to 1 indicates an additive effect between the drugs; and a CI greater than 1 indicates an antagonistic effect between the drugs.

[0062] The results are as follows Figure 8As shown in the figure. The results showed that in the CCRF-CEM cell line, the CI values ​​of 7.5 μM FSP1 inhibitor combined with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 0.5069, 0.5703, and 0.6311, respectively, all less than 1; in the MOLT-4 cell line, the CI values ​​of 7.5 μM FSP1 inhibitor combined with 0.2 nM, 0.5 nM, and 1 nM Ara-C were 0.4429, 0.5421, and 0.6689, respectively, all less than 1, indicating a synergistic effect between the FSP1 inhibitor and low-dose Ara-C.

[0063] Example 4 Detection of splenic and bone marrow infiltration by chemotherapy drugs in a non-ETP ALL mouse model 1. Five- to six-week-old female NOD / SCID mice were injected with GFP via the tail vein. + CCRF-CEM cells (2×10⁻⁶) 6 A non-ETP ALL mouse model was constructed using 25 mice (n=10 cells / mouse). On day 11 post-injection, mice were randomly divided into four groups: a saline or DMSO control group (n=7), an Ara-C monotherapy group (n=6), an FSP1 inhibitor monotherapy group (n=6), and an Ara-C + FSP1 inhibitor combination therapy group (n=6). Low-dose Ara-C and / or FSP1 inhibitors were injected intraperitoneally on days 11, 13, 15, 17, 19, 21, and 23, respectively; the Ara-C dose was 30 mg / kg, and the FSP1 inhibitor dose was 5 mg / kg. Mice were sacrificed on day 27 (4 days after injection). The administration strategy described above is as follows: Figure 9 As shown.

[0064] 2. The mice from step 1 of Example 4 were euthanized by cervical dislocation, and their spleens and bone marrow were further separated. The separated spleens were arranged according to the following groups: the first row was the saline and DMSO control group, with #1-#4 being the saline group and #5-#7 being the DMSO group from left to right; the second row was the Ara-C monotherapy group; the third row was the FSP1 inhibitor monotherapy group; and the fourth row was the Ara-C + FSP1 inhibitor combination therapy group. A ruler was placed below the groups.

[0065] Spleens in each group as follows Figure 10 As shown in the figure. The results showed that compared with the saline or DMSO control group, the spleens of mice in the Ara-C monotherapy group, the FSP1 inhibitor monotherapy group, and the Ara-C+FSP1 inhibitor combination therapy group were significantly smaller; compared with the Ara-C monotherapy group, the spleens of mice in the Ara-C+FSP1 inhibitor combination therapy group were further smaller.

[0066] 3. Take the mouse spleen and bone marrow from step 2 of Example 4, and grind them into a single-cell suspension. Flow cytometry was used to detect GFP levels in the spleen and bone marrow cells of mice from different drug treatment groups. + Cell percentage, reflecting the extent to which leukemia cells infiltrate the spleen and bone marrow.

[0067] Representative flow cytometry analysis diagrams are as follows: Figure 11 As shown, the statistical results are as follows: Figure 12 As shown in the figure. Results showed that in the mouse spleen, the percentage of leukemia cell infiltration was 7.73% ± 1.67% in the saline or DMSO control group; 5.91% ± 1.73% in the Ara-C monotherapy group; and 4.02% ± 0.9% in the Ara-C + FSP1 inhibitor combination therapy group. In the mouse bone marrow, the percentage of leukemia cell infiltration was 10.62% ± 3.8% in the saline or DMSO control group; 1.66% ± 1.03% in the Ara-C monotherapy group; and only 0.42% ± 0.46% in the Ara-C + FSP1 inhibitor combination therapy group. These results indicate that low-dose Ara-C combined with an FSP1 inhibitor can significantly reduce the infiltration of leukemia cells in the mouse spleen and bone marrow.

[0068] As can be seen from the above, the combination of FSP1 inhibitor and Ara-C not only synergistically increases the sensitivity of non-ETP ALL cells to low-dose Ara-C, but also significantly reduces the infiltration of leukemia cells in the spleen and bone marrow of mice.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents or methods for detecting the expression level of FSP1 protein or the gene encoding FSP1 protein in the preparation of products for prognostic assessment of non-early precursor T-cell acute lymphoblastic leukemia; The amino acid sequence of the FSP1 protein is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the FSP1 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The reagents for detecting the expression level of FSP1 protein include antibodies that specifically bind to FSP1; the methods for detecting the expression level of FSP1 protein include Western blotting.

3. The application according to claim 1, characterized in that, The prognostic indicators include overall survival and / or event-free survival.

4. Application of FSP1 inhibitors in the preparation of drugs for the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

5. Application of FSP1 inhibitors in the preparation of drugs that enhance the sensitivity of cytarabine to the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

6. Application of FSP1 inhibitors in combination with cytarabine in the preparation of drugs for the treatment of non-early precursor T-cell acute lymphoblastic leukemia.

7. The application according to any one of claims 4 to 6, characterized in that, The FSP1 inhibitor includes at least one of the following: 1) Substances that inhibit the activity of FSP1 protein; 2) Substances that reduce FSP1 protein levels; 3) Silencing, knockout, or mutation AIFM2 Genetic material; 4) Interference or suppression AIFM2 Substances involved in gene expression.

8. The application according to claim 7, characterized in that, The substances that inhibit the activity of FSP1 protein include FSP1 antibodies, peptides, or small molecule compounds; The substances that reduce FSP1 protein content include substances that promote FSP1 protein degradation.

9. The application according to any one of claims 4 to 6, characterized in that, The drug is in the form of an injection.

10. A medicament for treating non-early precursor T-cell acute lymphoblastic leukemia, characterized in that, The active ingredients include FSP1 inhibitors and cytarabine.

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