CN116179705A

CN116179705A - Application of SREBF1 in preparing chemotherapy medicament for treating acute T lymphocyte leukemia

Info

Publication number: CN116179705A
Application number: CN202310141915.3A
Authority: CN
Inventors: 纪春岩; 叶静静; 纪敏; 孙涛; 贾睿楠
Original assignee: Qilu Hospital of Shandong University
Current assignee: Qilu Hospital of Shandong University
Priority date: 2023-02-17
Filing date: 2023-02-17
Publication date: 2023-05-30

Abstract

The invention discloses an application of SREBF1 in preparing a chemotherapeutic medicament for treating acute T lymphocyte leukemia, and belongs to the technical field of biological medicines. The invention clearly shows the change of bone marrow microenvironment of a patient with T-ALL before and after chemotherapy and inhibits the protection effect of the patient on tiny residual diseases, and clearly shows the effect of SREBF1 in abnormal lipidation of bone marrow microenvironment after T-ALL chemotherapy, and the inhibition of the function of SREBF1 can inhibit the generation of fat cells induced by DEX, so as to reverse the protection effect of fat cells on residual T-ALL cells. Therefore, the inhibition of SREBF1 is a possible strategy for solving the T-ALL recurrence dilemma, is beneficial to the development of clinical treatment medicaments for eliminating the T-ALL tiny residue, provides new treatment ideas and medicaments for effectively eliminating the T-ALL tiny residue diseases and further improves the treatment of the T-ALL, and has very important clinical significance.

Description

Application of SREBF1 in preparing chemotherapy medicament for treating acute T lymphocyte leukemia

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of SREBF1 in preparation of a chemotherapeutic medicament for treating acute T-lymphocyte leukemia.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Acute T-lymphoblastic leukemia (T-acute lymphoblastic leukemia, T-ALL) is an invasive hematological malignancy caused by clonal expansion of variant T-cell precursors, accounting for about 25% of ALL adult cases. The use of intensive combination chemotherapy (at least one glucocorticoid, vincristine and an anthracycline) and the adjustment of the treatment regime according to the patient's response, the treatment of T-ALL makes a great progress, the complete remission rate is very high, but relapse also frequently occurs, and most patients are refractory and relapse, eventually leading to treatment failure. Five-year survival rates for adults under 60 years of age are only 40% -50%, with older patients having a poorer prognosis. The root cause of the recurrence is the presence of minimal residual disease (minimal residual disease, MRD), while the bone marrow microenvironment (Bone marrow microenvirent, BMM) is the main site of MRD, shielding residual leukemia cells from chemotherapy drugs.

Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) and adipocytes, endothelial cells, osteoblasts, fibroblasts and the like together form bone marrow stromal cells, and are the main components of the bone marrow microenvironment. Recent studies have shown that bone marrow adipocytes can participate in the development of malignant tumors by providing energy to solid tumor cells; in multiple myeloma, bone marrow adipocytes protect multiple myeloma cells from chemotherapy-induced apoptosis by secreting derived adipokine and promote bone metastasis. Adipocytes also play an important role in leukemia survival, proliferation, invasion and resistance.

Bone marrow mesenchymal stem cells (BMSCs) are precursor cells to adipocytes, and some chemotherapeutics can promote differentiation of BMSCs toward adipocytes. For example, in acute myeloid leukemia, the self-renewal capacity of bone marrow mesenchymal stem cells is reduced, and after treatment with cytarabine (Ara-C), they are easily differentiated into adipocytes and chondrocytes. However, the change condition of bone marrow microenvironment adipocytes of a patient with T-ALL after chemotherapy is not reported, and the molecular mechanism involved in regulating and controlling the change of bone marrow microenvironment of the patient with T-ALL before and after chemotherapy is not clear.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides application of SREBF1 in preparing a chemotherapeutic medicament for treating acute T lymphocyte leukemia. The invention aims to clarify the change of bone marrow microenvironment of a patient with T-ALL before and after chemotherapy and inhibit the protection effect of the patient on tiny residual diseases, and provides a new treatment idea and medicament for effectively eliminating the tiny residual diseases of the patient with T-ALL and further improving the treatment of the patient with T-ALL.

The present invention provides the following means based on the findings in view of the above-described state of the art.

In a first aspect of the invention, there is provided the use of a substance for detecting the expression level of SREBF1 in the preparation of a product for assessing prognosis of a patient with acute T-lymphoblastic leukemia.

The present invention has found through research that the expression level of SREBF1 in the BMSCs group after the induction of adipogenic differentiation is significantly increased compared with the BMSCs group without adipogenic differentiation.

In a second aspect of the invention, there is provided a product for assessing the prognosis of acute T-lymphocyte leukemia, said product comprising a substance for detecting SREBF 1.

Among these, substances that detect SREBF1 include, but are not limited to, substances used for RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing to detect the expression level of SREBF 1.

Such products include, but are not limited to, primers, probes, chips, nucleic acid membrane strips, formulations or kits for detecting the expression level of SREBF1 in a test sample.

The sample to be tested may be a human sample, more specifically, the sample to be tested includes spleen, or bone marrow of a subject.

In a third aspect of the invention, there is provided a system for diagnosing or aiding in the diagnosis of recurrence of acute T-lymphocyte leukemia, the system comprising:

i) An analysis unit comprising: a test substance for determining the expression level of SREBF1 in a test sample of a subject, and;

ii) an evaluation unit comprising: determining whether acute T lymphocyte leukemia in said subject recurs based on said SREBF1 expression level determined in i);

the step ii) the specific evaluation flow of the evaluation unit comprises the following steps:

the subject is or is candidate as a patient with recurrent acute T-lymphoblastic leukemia, if the level of SREBF1 expression in the test sample of the subject is up-regulated compared to the reference; conversely, the subject is not or is not candidate for a patient with a relapse of acute T-lymphocyte leukemia.

In a fourth aspect, the invention provides an application of SREBF1 as a treatment target of abnormal bone marrow microenvironment lipidation after acute T-lymphocyte leukemia chemotherapy in preparing a medicament for treating acute T-lymphocyte leukemia.

In a fifth aspect, the invention provides the use of a substance that inhibits SREBF1 expression and/or reduces its activity in the preparation of a product for combating abnormal bone marrow microenvironment lipidation following acute T-lymphoblastic leukemia chemotherapy.

In a sixth aspect, the invention provides the use of a substance that inhibits SREBF1 expression and/or reduces its activity in the manufacture of a medicament for inhibiting acute T-lymphoblastic leukemia relapse.

The beneficial effects of the invention are as follows:

the invention clearly shows the change of bone marrow microenvironment of a patient with T-ALL before and after chemotherapy and inhibits the protection effect of the patient on tiny residual diseases, and clearly shows the effect of SREBF1 in abnormal lipidation of bone marrow microenvironment after T-ALL chemotherapy, and the inhibition of the function of SREBF1 can inhibit the generation of fat cells induced by DEX, so as to reverse the protection effect of fat cells on residual T-ALL cells. Inhibition of SREBF1 may therefore be a viable strategy to address the dilemma of T-ALL recurrence.

The invention provides a new insight for survival mechanism of residual T-ALL cells after chemotherapy from the aspect of improving bone marrow microenvironment, is beneficial to the development of clinical treatment medicaments for eliminating the residual T-ALL, provides a new treatment idea and medicament for effectively eliminating the residual T-ALL, and further improves the treatment of the T-ALL, and has very important clinical significance.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 shows the proportion of adipocytes in the bone marrow microenvironment before and after chemotherapy of a primary T-ALL patient by H & E staining; wherein, the Diagnosis is: bone marrow biopsy specimens of T-ALL primary patients; post-chemo is: complete remission of the bone marrow biopsy specimens of the T-ALL patients after chemotherapy corresponding to the initial diagnosis;

FIG. 2 shows the effect of BODIPY fluorescent staining on BMSCs adipogenic differentiation by chemotherapeutic agents;

FIG. 3 is a graph showing the effect of adipocytes on T-ALL cell adhesion, anti-apoptotic capacity, and colony forming capacity;

FIG. 4 is a molecular screen for differential expression during adipogenic differentiation of BMSCs by RNA-Seq in example 1;

FIG. 5 is an enrichment analysis of differentially expressed molecules during adipogenic differentiation of BMSCs in example 1;

FIG. 6 shows the change in SREBF1 expression level during adipogenic differentiation of BMSCs in example 1;

FIG. 7 is an in vitro experiment in example 2 demonstrating the effect of inhibiting the function of SREBF1 on adipogenic differentiation of BMSCs;

FIG. 8 is an in vivo experiment in example 3 demonstrating the effect of inhibiting the function of SREBF1 on adipogenic differentiation of BMSCs;

FIG. 9 is an in vitro validation of the effect of reversing adipocyte protection with SREBF1 inhibitors in example 4;

fig. 10 is an in vivo validation of the effect of reversing adipocyte protection with SREBF1 inhibitors in example 5.

Detailed Description

The invention will be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not to be construed as limiting the invention. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the sales company; materials, reagents and the like used in the examples were commercially available unless otherwise specified.

The term "expression level" refers to the amount of a gene product present in vivo or in a sample at a particular point in time. The expression level can be measured/quantified/detected, for example, by the protein or mRNA expressed by the gene. Expression levels can be quantified, for example, as follows: the amount of the gene product of interest present in the sample is normalized with the total amount (total protein or mRNA) of the same type of gene product in the same sample or reference sample (e.g., a sample obtained from the same individual at the same time or a portion of the same sample of the same size (weight, volume), or the amount of the gene product of interest/defined sample size (weight, volume, etc.) is determined. The expression level may be measured or detected by any method known in the art, such as methods for direct detection and quantification of a gene product of interest (e.g., mass spectrometry), or methods for indirect detection and measurement of a gene product of interest that typically work by binding the gene product of interest to one or more different molecules or detection devices (e.g., primers, probes, antibodies, protein scaffolds) that are specific for the gene product of interest. It is also known to the skilled person to determine the level of gene copies, which also includes determining the absence or presence of one or more fragments (e.g. by nucleic acid probes or primers, such as quantitative PCR, multiplex ligation dependent probe amplification (Multiplex ligation-dependent probe amplification, MLPA) PCR).

The terms "index" and "marker" are used interchangeably herein and refer to a sign or signal of a condition or for monitoring a condition. Such "disorder" refers to a biological state of a cell, tissue or organ, or to a health and/or disease state of an individual. The indicator may be the presence or absence of a molecule including, but not limited to, a peptide, protein, and nucleic acid, or may be a change in the level or pattern of expression of such a molecule in a cell, or tissue, organ, or individual. The indicator may be the occurrence, development or presence of a disease in an individual or a sign of further progression of such a disease. The indicator may also be a sign of the risk of developing a disease in the individual.

The terms "up-regulation", "elevation" or "elevation" of the level of an indicator refer to a decrease in the level of such an indicator in a sample as compared to a reference.

The terms "down-regulation", "decrease" or "decrease" in the level of an indicator refer to a decrease in the level of such an indicator in a sample as compared to a reference.

The term "kit" as used herein refers to a collection of the above components, preferably provided separately or in a single container. The container also preferably contains instructions for carrying out the method of the invention. Examples of these components of the kit and methods of use thereof have been given in this specification. Preferably, the kit comprises the above components in a ready-to-use formulation. Preferably, the kit may additionally comprise instructions, for example a user manual for adjusting the components (e.g. the concentration of the detection agent) and for interpreting the results of any assays regarding the diagnosis provided by the method of the invention. In particular, such a manual may include information for assigning the determined amount of gene product to the diagnostic type. Details are found elsewhere in this specification. Further, such a user manual may provide instructions for proper use of the kit components for determining the amount of the corresponding biomarker. The user manual may be provided in paper or electronic form (e.g., stored on a CD or CD ROM). The invention also relates to the use of said kit in any method according to the invention.

The term "system" as used herein relates to a device system comprising at least the above devices that are effectively interconnected to allow diagnosis. Preferred means for determining the methylation status or amount of a gene product and means for performing a comparison are disclosed above in connection with the methods of the invention. How the devices are operatively connected will depend on the type of device included in the apparatus. For example, in the case of the application of a device for the automated determination of the methylation state or amount of a gene product, the data obtained by the automated handling device can be processed by, for example, a computer program to establish a diagnosis. Preferably, in this case, the apparatus is contained in a single device. Thus, the device may comprise an analysis unit for determining the methylation status or amount of the gene product in the sample and an evaluation unit for processing the resulting data for diagnosis. Preferred detection devices are disclosed above in connection with embodiments relating to the methods of the present invention. In this case, the device is operatively connected so that the user of the system, thanks to the instructions and explanations given in the manual, combines the determination of the quantity and its diagnostic value. In such embodiments the device may be presented as a separate apparatus and preferably packaged together as a kit. Those skilled in the art will appreciate how to contact the device without further inventive skill. Preferred devices are those that can be applied without the specific knowledge of a skilled clinician, such as test strips or electronic devices that need only to be loaded with a sample. The result may be given as an output of parametric diagnostic raw data, preferably as an absolute or relative quantity. It should be appreciated that these data will need to be interpreted by a clinician. However, expert system devices are also contemplated in which the output contains processed diagnostic raw data, the interpretation of which does not require a specialized clinician. Further preferred devices comprise an analytical unit/device (e.g. biosensor, array, solid support coupled to a ligand that specifically recognizes a polypeptide, plasmon surface resonance device, NMR spectrometer, mass spectrometer, etc.) or an evaluation unit/device as mentioned above in accordance with the methods of the invention.

As described in the background art, aiming at the phenomenon that the acute T lymphocyte leukemia recurs due to the current T-ALL minimal residual disease, the invention aims to provide the application of SREBF1 in preparing the chemotherapeutic drugs for treating the acute T lymphocyte leukemia, define the change of the bone marrow microenvironment after chemotherapy and further shelter the mechanism of residual T-ALL cells, and provide a new therapeutic target for treating the minimal residual disease.

In an exemplary embodiment of the invention, the use of a substance that detects the expression level of SREBF1 for the preparation of a product for assessing prognosis of a patient with acute T-lymphoblastic leukemia.

Wherein the SREBF1 is up-regulated in the expression of patients with recurrent acute T-lymphoblastic leukemia following chemotherapy.

The invention applies the bone marrow biopsy tissues corresponding to the treatment of the adult T-ALL patient one by one, compares the pathological changes of the bone marrow biopsy before and after the chemotherapy of the T-ALL patient, and discovers that the fat cells in the bone marrow are obviously increased after the chemotherapy of the T-ALL patient; further studies have found that BMSCs cultured in vitro in T-ALL patients promote their differentiation into adipocytes upon addition of the chemotherapeutic drug Dexamethasone (DEX). And further confirmed that BMSCs-derived adipocytes have supporting and protective effects on residual T-ALL cells. Meanwhile, the key molecule SREBF1 of DEX induced BMSCs abnormal adipogenic differentiation is screened out through RNA-Seq, and the subsequent in vitro cell experiment and in vivo animal experiment prove that inhibiting SREBF1 can reduce the promotion effect of DEX on BMSCs adipogenic differentiation and inhibit the protection effect of adipocytes on residual T-ALL cells for the first time. Therefore, SREBF1 can be used as one of targets of the T-ALL minimal residual disease. The present invention has found through research that the expression level of SREBF1 in the BMSCs group after the induction of adipogenic differentiation is significantly increased compared with the BMSCs group without adipogenic differentiation.

In yet another embodiment of the invention, a product is provided comprising the above-described substance for detecting SREBF1 for use in assessing acute T-lymphocyte leukemia prognosis.

The sample to be tested is a human sample and a non-human sample, more specifically, the sample to be tested comprises cells, tissues, organs and body fluids of a subject.

Wherein the tissue may be spleen and bone marrow;

still further, in addition to the SREBF1 markers described above, the products include substances suitable for detecting other biomarkers of acute T-lymphocyte leukemia recurrence, including, but not limited to, endocrine factor related biomarkers, infective factor related biomarkers (such as any severe infection that can cause bacteremia or viremia), immune function related biomarkers (such as anti-phospholipid antibodies, anti-nuclear antibodies, anti-DNA antibodies, anti-sperm antibodies, anti-thyroid antibodies, natural Killer (NK) cell numbers and activity elevation, macrophage dysfunction, dendritic cell dysfunction, complement system dysfunction, blocking antibody deficiency, T, B lymphocyte abnormality, helper T-lymphocyte (Th 1/Th2 cytokine abnormality, etc.), pre-thrombotic state related biomarkers (such as factor V and factor II (thrombopoietin) gene mutations, protein S deficiency), and other systemic diseases in a subject. By combining with other biomarkers, the interference of other physiological and pathological states is further eliminated, and the sensitivity and specificity of detection are further improved.

In yet another embodiment of the present invention, there is provided a system for diagnosing or aiding in the diagnosis of recurrence of acute T-lymphocyte leukemia, the system comprising:

ii) an evaluation unit comprising: determining whether acute T-lymphocyte leukemia in said subject recurs based on said SREBF1 expression level determined in i).

In yet another embodiment of the present invention, the above test sample comprises a subject cell, tissue, organ and body fluid;

wherein the tissue may be spleen or bone marrow.

In yet another embodiment of the present invention, the detection materials described above include, but are not limited to, materials used for RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing to detect the expression level of SREBF 1.

In yet another embodiment of the invention, further biomarkers suitable for detecting recurrence of acute T-lymphocyte leukemia are included in the assay unit, including but not limited to endocrine factor related biomarkers, infective factor related biomarkers (such as any severe infection that can cause bacteremia or viremia), immune function related biomarkers (such as anti-phospholipid antibodies, anti-nuclear antibodies, anti-DNA antibodies, anti-sperm antibodies, anti-thyroid antibodies, natural Killer (NK) cell numbers and activity elevation, macrophage dysfunction, dendritic cell dysfunction, complement system dysfunction, blocking antibody deficiency, T, B lymphocyte abnormality, helper T-lymphocyte (Th 1/Th2 cytokine abnormality, etc.), pre-thrombotic state related biomarkers (such as factor V and factor II (thrombopoietin) gene mutations, protein S deficiency), and other systemic diseases of the subject, etc. By combining with other biomarkers, the interference of other physiological and pathological states is further eliminated, and the sensitivity and specificity of detection are further improved.

In still another embodiment of the present invention, the specific evaluation procedure of the evaluation unit includes:

Where a "reference" may be a suitable control sample, e.g., a sample from a normal healthy subject that is free of symptoms associated with recurrent abortion and free of abnormal physiological and pathological findings, the reference may also be a sample from the same subject prior to the manifestation of symptoms of the disorder or disease or prior to the diagnosis of recurrent abortion. The reference may be a normalized sample, e.g., a sample comprising material or data from several healthy subject samples that are free of recurrent abortion symptoms, nor related physiological and pathological findings.

The system for diagnosing or aiding in diagnosing recurrent abortion of the present invention may be a virtual device as long as the functions of the analyzing unit and the evaluating unit can be realized. The analysis unit can comprise various detection reagent materials and/or detection instrument devices and the like; the evaluation unit may be any operation instrument, module or virtual device capable of analyzing the detection result of the analysis unit to obtain recurrent abortion disease risk evaluation status, for example, various possible detection results and corresponding disease risk conditions may be formulated into corresponding data charts in advance, and the detection result of the detection unit is compared with the data charts to obtain recurrent abortion disease risk evaluation results.

In still another embodiment of the present invention, there is provided an application of SREBF1 as a therapeutic target for treating abnormal bone marrow microenvironment after chemotherapy of acute T-lymphocyte leukemia in the preparation of a medicament for treating acute T-lymphocyte leukemia.

In yet another embodiment of the invention, the use comprises the use of a substance that inhibits SREBF1 expression and/or reduces its activity for the preparation of a chemotherapeutic agent for the treatment of acute T-lymphoblastic leukemia.

In yet another embodiment of the present invention, the substance that inhibits the expression and/or reduces the activity of SREBF1 may be an interfering molecule that targets SREBF1 and is capable of inhibiting the expression of SREBF1, and specifically may include shRNA (small hairpin RNA), small interfering RNA (siRNA), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming the shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid; inhibitors of the class of compounds may also be included, preferably including but not limited to Fatostatin HBr, shRNA knockdown SREBF 1.

The invention uses SREBF1 to down regulate slow virus (shSREBF 1) or SREBF1 inhibitor (Fastatin HBr, FH) to treat BMSCs of T-ALL patients. Inducing BMSCs to differentiate towards fat by using DEX, and obviously increasing the number of lipidated cells and lipid droplets of each group along with the extension of the induction time; DEX-induced lipidated cell numbers and lipid droplets were significantly reduced when either lentiviruses were used to down-regulate SREBF1 or SREBF1 inhibitors were used for dry. Wherein, the nucleotide sequence of the SREBF1 down-regulating lentivirus I (shSREBF 1) is as follows: TCTCCATCAGTTCCAGCAT.

In yet another embodiment of the present invention, the chemotherapeutic agent for treating acute T-lymphoblastic leukemia comprises an agent that inhibits T-ALL minimal residual disease.

In yet another embodiment of the invention, the use of a substance that inhibits SREBF1 expression and/or reduces its activity in the preparation of a product for the treatment of abnormal bone marrow microenvironment following chemotherapy for acute T-lymphoblastic leukemia.

Wherein the substance comprises Fatostatin HBr and shRNA for knocking down SREBF 1. The nucleotide sequence of the shRNA for knocking down SREBF1 is as follows: TCTCCATCAGTTCCAGCAT.

In yet another embodiment of the invention, the function of the product is any one or more of the following:

a. inhibiting glucocorticoid from inducing adipogenesis in bone marrow microenvironment;

b. inhibit the protective effect of fat cells on residual T-ALL cells in the bone marrow microenvironment.

In yet another embodiment of the present invention, the glucocorticoid comprises dexamethasone DEX.

In yet another embodiment of the invention, the product consists of a substance that inhibits SREBF1 expression and/or reduces its activity and pharmaceutically necessary excipients.

In yet another embodiment of the present invention, the product may be a pharmaceutical.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a blending agent, a surfactant, a dispersing agent, or an antifoaming agent.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a virus, a microcapsule, a liposome, a nanoparticle, or a polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier may be a liposome, biocompatible polymer (including natural and synthetic polymers), lipoprotein, multi-skin, polysaccharide, lipopolysaccharide, artificial viral envelope, inorganic (including metallic) particles, as well as bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid or plasmid vectors.

The medicament may also be used in combination with other medicaments for the prevention and/or treatment of recurrent abortion, and other prophylactic and/or therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The medicament may also be administered alone in separate compositions or in a dosage form different from the primary active ingredient, with other prophylactic and/or therapeutic compounds. A partial dose of the principal component may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the medicament of the invention may be adjusted during the course of treatment according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In yet another embodiment of the present invention, there is provided the use of a substance that inhibits SREBF1 expression and/or reduces its activity in the manufacture of a medicament for inhibiting acute T-lymphocyte leukemia relapse prevention.

In order to verify the feasibility of using a substance for inhibiting the expression and/or reducing the activity of SREBF1 as a drug for inhibiting the residual active ingredient after chemotherapy of acute T lymphocyte leukemia, the invention uses an NPG xenograft mouse model to carry out in vivo experiments, the proportion of DEX group is obviously reduced compared with Control group when peripheral blood is treated in the end of the 3 rd period, and the DEX group shows statistically significant treatment effect, but the DEX+SREBF1 inhibitor group has no obvious difference in treatment effect compared with the DEX group. At the end of treatment cycles 4 and 5, the proportion of the DEX group is significantly reduced compared with the Control group, and the DEX group has a more obvious treatment effect; the proportion of T-ALL cells in the combination group was significantly reduced in the DEX+SREBF1 inhibitor group compared to the DEX group. After the 5 th period of administration, the mice are euthanized, compared with the Control group, the size and weight of spleen of the DEX group are obviously reduced, and the proportion of T-ALL cells in spleen and bone marrow is also obviously reduced; compared with the DEX group, the size and weight of spleen of the combined administration group are obviously reduced, and the proportion of T-ALL cells in spleen and bone marrow is also obviously reduced. Substances which suggest that the expression of SREBF1 is inhibited and/or the activity of the SREBF1 is reduced can be used as medicines for inhibiting residual active ingredients after chemotherapy of acute T lymphocyte leukemia.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

EXAMPLE 1 DEX-induced changes in SREBF1 expression during adipogenic differentiation of BMSCs

RNA-seq analysis was performed using T-ALL samples, systematically exploring the differences in changes in BMSCs during adipogenic differentiation.

In vitro isolation 3 cases of BMSCs from T-ALL patients were cultured and treated in 2 groups:

(1) BMSC group (BMSCs): culturing BMSCs using alpha-MEM containing 10% fetal bovine serum;

(2) BMSC group (Induced-BMSCs) Induced for 14 days of adipogenic differentiation: the BMSCs were induced to adipogenic differentiation by addition of 10% fetal bovine serum alpha-MEM containing 0.5mM 3-isobutyl-1-methylxanthine (IBMX), 10. Mu.g/ml insulin, 1. Mu.M Dexamethasone (DEX) at a final concentration.

Cells were collected on day 14, RNA was extracted, and RNA-Seq detection was performed to screen genes differentially expressed before and after adipogenic differentiation of BMSCs.

The detection result shows that: there was a clear molecular difference between the BMSCs group and the BMSCs group after induction of adipogenic differentiation, a total of 3886 genes were significantly differentially expressed between the two groups (q <0.05,

log2fold-chang| >0, fig. 4), including 1813 up-regulated genes and 2073 down-regulated genes.

KEGG functional enrichment analysis was then performed on 1813 genes up-regulated in the BMSCs group after induction of adipogenic differentiation (fig. 5), and the results showed that these up-regulated genes were clearly involved in oxidative phosphorylation, fatty acid metabolism, biosynthesis of unsaturated fatty acids, etc., all of which are involved in fat formation.

Based on the functional enrichment results, 209 genes associated with adipogenesis were further selected. Wherein the expression of SREBF1 is significantly up-regulated in the induced BMSCs group, thus SREBF1 may be a key target for abnormal adipogenic differentiation of bone marrow BMSCs in T-ALL patients after chemotherapy.

The results of verifying the expression of SREBF1 in BMSCs after adipogenic differentiation induction by qRT-PCR and Western-Blot are shown in FIG. 6, and the expression level of SREBF1 in the BMSCs after adipogenic differentiation induction is obviously increased compared with that in the BMSCs group, consistent with the RNA-Seq result, and suggest that DEX can promote the differentiation of BMSCs to adipocytes by regulating the expression of SREBF 1.

Example 2 in vitro assay of the effects of inhibiting SREBF1 function on adipogenic differentiation of BMSCs

In vitro experiments, T-ALL patients were treated with SREBF1 downregulated lentivirus (shSREBF 1) or SREBF1 inhibitor (Fatostatin HBr, FH), BMSCs were induced to differentiate in the adipose direction with DEX, and the effect of intervening SREBF1 on DEX-induced adipogenic differentiation of BMSCs was examined on

days

7, 14, and 21 of induction with oil red O staining. Wherein, the nucleotide sequence of SREBF1 down-regulating lentivirus shSREBF1 is as follows: TCTCCATCAGTTCCAGCAT.

The results are shown in FIG. 7: with the extension of the induction time, the number of lipidated cells and lipid droplets of each group are obviously increased; DEX-induced lipidated cell numbers and lipid droplets were significantly reduced when either lentiviruses were used to down-regulate SREBF1 or SREBF1 inhibitors were used for dry. It was suggested that inhibition of expression or action of SREBF1 could reverse the promoting effect of DEX on differentiation of BMSCs toward adipocytes, suggesting that SREBF1 plays a key role in the process of DEX inducing differentiation of BMSCs toward adipocytes.

Example 3 in vivo detection of the Effect of SREBF1 inhibition of function on adipogenic differentiation of BMSCs in mice

In vivo experiments, firstly, transfecting a human T-ALL cell line Jurkat cell by using an empty lentivirus carrying Green Fluorescent Protein (GFP), carrying out flow separation on GFP+ cells, then carrying out amplification culture, selecting severe immunodeficiency mice of 6-8 weeks old, radiating with 1.5Gy radiation, inoculating GFP+ Jurkat cells into each mouse body by tail vein injection, establishing a T-ALL human xenograft model, continuously monitoring the content of GFP+ cells in peripheral blood of the mice by using flow cytometry, dividing the mice into 3 groups when the proportion of GFP+ cells in the peripheral blood can be monitored, and starting administration.

A first group: control group, given Control solvent treatment;

a second group, DEX group, given 1.5mg/kg/d DEX treatment;

third group: DEX+SREBF1 inhibitor group, 15mg/kg/dSREBF1 inhibitor (FatostatinHBr (FH)) in combination with 1.5mg/kg/d DEX.

The administration is continued for 5 days and 2 days for a period of 5 periods for 35 days.

Selecting mice after the 3 rd period and the 5 th period, extracting femur of the mice, performing section staining, and observing the content of fat cells.

As a result, as shown in fig. 8, the fat content of the DEX group was significantly higher than that of the Control group and the dex+srebf1 inhibitor group, compared to the Control group. The bone marrow fat content of the DEX group at the end of cycle 5 was significantly higher than that of the DEX group at the end of cycle 3. Demonstrating that DEX promotes adipogenic differentiation of T-ALL mouse bone marrow BMSCs during treatment, and that application of SREBF1 inhibitors can block DEX-induced adipogenic differentiation of mouse bone marrow BMSCs.

Example 4 in vitro validation of the effects of application of SREBF1 inhibitors to reverse the adipocyte protection

Continuously intervening in the fat differentiation process of BMSCs by using an SREBF1 inhibitor (Fatostatin HBr), inducing the BMSCs to undergo fat differentiation for 21 days, and directly co-culturing the fat cells or the fat cells inhibited by the SREBF1 and the T-ALL cells by taking a fat cell group which is not treated by the inhibitor as a control, wherein after the SREBF1 inhibitor is used, the adhesion to the T-ALL cells is obviously reduced along with the reduction of the fat differentiation proportion of the BMSCs; the apoptosis rate induced by the T-ALL cell drug is obviously increased, and the proportion of the living cells is obviously reduced; the colony forming ability of T-ALL cells was significantly reduced. (FIG. 9)

EXAMPLE 5 use of SREBF1 inhibitors to reverse the Effect of T-ALL mice on adipocyte protection after chemotherapy

According to the mouse model in example 3, peripheral blood at the end of treatment cycle 3, the proportion of gfp+ cells was significantly reduced in the DEX group compared to the Control group, and the DEX group exhibited statistically significant therapeutic effects, but there was no significant difference in the dex+srebf1 inhibitor group compared to the DEX group.

At the end of treatment cycles 4 and 5, the proportion of GFP+ cells was significantly reduced in the DEX group compared to the Control group, the DEX group having a more pronounced therapeutic effect; the proportion of gfp+ cells in the combination group was significantly reduced in the dex+srebf1 inhibitor group compared to the DEX group.

After the 5 th period of administration, the mice are euthanized, compared with the Control group, the size and weight of spleen of the DEX group are obviously reduced, and the proportion of T-ALL cells in spleen and bone marrow is also obviously reduced;

compared with the DEX group, the size and weight of spleen of the combined administration group are obviously reduced, and the proportion of T-ALL cells in spleen and bone marrow is also obviously reduced. (FIG. 10)

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a substance that detects the expression level of SREBF1 for the preparation of a product for assessing prognosis of a patient with acute T-lymphoblastic leukemia.

2. The use of claim 1, wherein SREBF1 is up-regulated in patients with recurrent acute T-lymphoblastic leukemia following chemotherapy.

3. A product for assessing the prognosis of acute T-lymphocyte leukemia, said product comprising a substance for detecting SREBF 1.

4. The product of claim 3, wherein the product comprises a primer, probe, chip, nucleic acid membrane strip, formulation, or kit for detecting the level of SREBF1 expression in a test sample;

preferably, the sample to be tested is a human sample and a non-human sample, more specifically, the sample to be tested comprises cells, tissues, organs and body fluids of a subject;

preferably, the tissue is spleen, bone marrow;

preferably, the product further comprises a substance suitable for detecting other biomarkers of acute T-lymphocyte leukemia recurrence, including endocrine factor related biomarkers, infective factor related biomarkers, immune function related biomarkers, pre-thrombotic state related biomarkers, and other systemic diseases in a subject;

preferably, the substance for detecting SREBF1 comprises a substance for detecting the expression level of SREBF1 by RT-PCR, real-time quantitative PCR, in situ hybridization, gene chip and gene sequencing.

5. A system for diagnosing or aiding in the diagnosis of recurrence of acute T-lymphocyte leukemia, the system comprising:

preferably, the analysis unit further comprises a substance suitable for detecting other biomarkers of acute T-lymphocyte leukemia recurrence, including genetic factor related biomarkers, endocrine factor related biomarkers, infective factor related biomarkers, immune function related biomarkers, pre-thrombotic status related biomarkers, and other systemic diseases of the subject;

preferably, the specific evaluation flow of the evaluation unit includes:

Application of SREBF1 as a treatment target of abnormal bone marrow microenvironment lipidation after acute T-lymphoblastic leukemia chemotherapy in preparing a chemotherapeutic medicament for treating acute T-lymphoblastic leukemia.

Preferably, the use comprises the use of a substance that inhibits SREBF1 expression and/or reduces its activity for the preparation of a chemotherapeutic agent for the treatment of acute T-lymphoblastic leukemia;

preferably, the substance comprises Fastatin HBr, shRNA knockdown SREBF 1;

preferably, the shRNA knocked down SREBF1 has a nucleotide sequence of:

TCTCCATCAGTTCCAGCAT。

7. application of a substance for inhibiting SREBF1 expression and/or reducing activity thereof in preparing a bone marrow microenvironment abnormal lipidation product after chemotherapy for resisting acute T lymphocyte leukemia;

preferably, the substance comprises Fastatin HBr, shRNA knockdown SREBF 1;

preferably, the shRNA knocked down SREBF1 has a nucleotide sequence of:

TCTCCATCAGTTCCAGCAT。

8. the use of claim 7, wherein the function of the product is any one or more of:

b. inhibiting the protective effect of fat cells in the bone marrow microenvironment on residual T-ALL cells;

preferably, the glucocorticoid comprises dexamethasone DEX.

9. The use according to claim 7, wherein the product consists of a substance which inhibits SREBF1 expression and/or reduces its activity and pharmaceutically necessary excipients.

10. Application of a substance for inhibiting SREBF1 expression and/or reducing activity thereof in preparing a medicament for inhibiting relapse prevention of acute T lymphocyte leukemia;

preferably, the substance comprises Fastatin HBr, shRNA knockdown SREBF 1;

preferably, the shRNA knocked down SREBF1 has a nucleotide sequence of:

TCTCCATCAGTTCCAGCAT。