Application of ACLY in regulating lipid metabolism and promoting drug sensitivity of PI3K inhibitors in chronic lymphocytic leukemia

Abstract

The present invention belongs to the field of biomedicine and molecular biology technology, and specifically relates to the application of ACLY in regulating lipid metabolism in chronic lymphocytic leukemia and promoting drug sensitivity of PI3K inhibitors. Studies have shown abnormal lipid accumulation in chronic lymphocytic leukemia cells and overexpression of ACLY, a key enzyme for fatty acid synthesis, in chronic lymphocytic leukemia, and serves as a hub for lipids, cholesterol and acetylation in chronic lymphocytic leukemia. At the same time, ACLY can be regulated by the PI3K‑AKT pathway and increase the drug sensitivity of Idelalisib. In summary, the research results of the present invention show that ACLY acts as a hub connecting cell metabolic epigenetics in chronic lymphocytic leukemia, and reveals the molecular mechanism of chronic lymphocytic leukemia cell proliferation, which helps to improve the treatment effect and therefore has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and molecular biology, and particularly relates to the application of ACLY in regulating lipid metabolism in chronic lymphocytic leukemia and promoting drug sensitivity of PI3K inhibitors. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Metabolic alterations have become one of the hallmarks of cancer. Recent advances have shown that the "Warburg dogma," which favors glycolysis over mitochondrial oxidative phosphorylation (OXPHOS) to provide cellular energy, does not actually apply to all cancers. Indolent hematological malignancies, in particular, can deviate from this paradigm; however, OXPHOS remains quite active in these cancers. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal B cells. A previous study showed that CLL cells in resting peripheral blood have higher OXPHOS than healthy B cells, but not higher levels of glycolysis. Currently, the use of targeted drugs has transformed the treatment of CLL and the prognosis of patients, but side effects and drug resistance still exist. Most patients with previously treated CLL relapse after first-line treatment and receive subsequent treatment.

[0004] Previous studies have shown that metabolic changes exist in chronic lymphocytic leukemia cells. In vitro studies have shown that tumor microenvironment (TME) signals can promote metabolic changes in chronic lymphocytic leukemia cells. ATP-citrate lyase (ACLY) catalyzes the Mg-ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate (OAA) and acetyl-CoA and is one of the key metabolic enzymes. ACLY is mainly located in the cytoplasm and nucleus and is associated with hepatic steatosis, dyslipidemia, diabetes, and many malignancies, such as colorectal cancer, breast cancer, prostate cancer, liver cancer, gastric cancer, cervical cancer, osteosarcoma, and glioblastoma.

[0005] Acetyl-CoA is a central metabolic intermediate produced by ACLY. It is a cornerstone of lipid synthesis and a substrate for the acetylation of proteins and metabolites. ACLY establishes a link between the catabolism of nutrients (glucose, glutamine and fatty acids), the biosynthesis required for DNA transcription and replication, and protein acetylation. Therefore, ACLY plays a key role in cancer metabolism by potentially depriving cytosolic citrate, promoting glycolysis and signaling pathways that support cancer development. ACLY can physically interact with the catalytic subunit AMPK, thereby inhibiting AMPK activity. ACLY knockdown can activate AMPK, leading to p53 activation, and ultimately leading to cellular senescence or DNA damage-induced cell death.

[0006] In addition, ACLY can be regulated by phosphorylation of the PI3K-AKT pathway, causing metabolic changes. Under physiological conditions, the PI3K-AKT pathway is activated by insulin, growth factors, and cytokines, and regulates key metabolic processes such as glucose metabolism, macromolecular biosynthesis, and maintenance of redox balance, supporting systemic metabolic homeostasis and the growth metabolism of individual cells. Oncogenic activation of the PI3K-AKT pathway in cancer cells reprograms cellular metabolism by increasing the activity of nutrient transporters and metabolic enzymes, thereby supporting the anabolic needs of abnormally growing cells. Understanding how the PI3K-AKT pathway controls metabolic networks in normal cells and how this control is altered in cancer cells can reveal metabolic vulnerabilities and provide information for new therapeutic strategies. Summary of the Invention

[0007] Based on the above-mentioned prior art, the inventors, after long-term technical and practical exploration, provide the application of ACLY in regulating lipid metabolism in chronic lymphocytic leukemia and promoting drug sensitivity to PI3K inhibitors. Specifically, the present invention provides new insights into metabolic reprogramming by examining the metabolic and acetylation status of chronic lymphocytic leukemia. Further characterization of the interaction between metabolism and acetylation revealed the key role of ACLY in chronic lymphocytic leukemia. In addition, in vitro studies have shown that the targeted inhibitor BMS-303141 has potential anti-tumor effects in chronic lymphocytic leukemia and can increase drug sensitivity to PI3K inhibitors. Based on the above research results, the present invention was completed.

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a reagent for detecting the expression level of ATP-citrate lyase for use in preparing a chronic lymphocytic leukemia assessment product. Specifically, the present invention has discovered through research that, compared with normal lymphocytes, lipid levels in chronic lymphocytic leukemia cell lines and cells from chronic lymphocytic leukemia patients are significantly elevated, and ACLY is overexpressed in chronic lymphocytic leukemia patients. Furthermore, chronic lymphocytic leukemia patients with higher ACLY expression have a shorter overall survival (OS), indicating that ACLY overexpression is associated with lipogenesis and poor prognosis in chronic lymphocytic leukemia.

[0010] Therefore, the chronic lymphocytic leukemia assessment product specifically assesses the adipogenesis and / or prognosis of chronic lymphocytic leukemia.

[0011] The product may be a test kit, a test device and / or an apparatus.

[0012] A second aspect of the present invention provides a system for evaluating chronic lymphocytic leukemia, the system comprising:

[0013] i) an analysis unit, the analysis unit comprising: a detection reagent for determining the expression level of the above-mentioned ATP-citrate lyase in a test sample of a subject, and;

[0014] ii) an evaluation unit, comprising: evaluating the disease condition of the subject according to the expression level of the ATP-citrate lyase determined in i).

[0015] The third aspect of the present invention provides the use of ATP-citrate lyase as a target in the preparation and / or screening of chronic lymphocytic leukemia drugs.

[0016] The chronic lymphocytic leukemia drug includes drugs for preventing and / or treating chronic lymphocytic leukemia.

[0017] A fourth aspect of the present invention provides the use of an ATP-citrate lyase inhibitor in any one or more of the following:

[0018] (a) preparing products for inhibiting the proliferation of chronic lymphocytic leukemia cells and / or promoting apoptosis and cell cycle arrest of chronic lymphocytic leukemia cells;

[0019] (b) preparing a product for increasing the sensitivity of chronic lymphocytic leukemia to PI3K inhibitors;

[0020] (c) Preparation of anti-chronic lymphocytic leukemia products.

[0021] A fifth aspect of the present invention provides the use of an ATP-citrate lyase inhibitor combined with a PI3K inhibitor in the preparation of a chronic lymphocytic leukemia drug.

[0022] Wherein, the ATP-citrate lyase may be BMS-303141, and the PI3K inhibitor may be Idelalisib.

[0023] A sixth aspect of the present invention provides a pharmaceutical composition, wherein the active ingredients of the pharmaceutical composition include at least an ATP-citrate lyase inhibitor and a PI3K inhibitor.

[0024] The active ingredients of the composition include at least BMS-303141 and idelalisib. The combination of the two can significantly inhibit the proliferation of chronic lymphocytic leukemia cells and reduce the survival rate of chronic lymphocytic leukemia cells, showing significant drug synergistic effect.

[0025] A seventh aspect of the present invention provides a method for treating chronic lymphocytic leukemia, comprising administering the above-mentioned ATP-citrate lyase inhibitor or the above-mentioned pharmaceutical composition to a patient.

[0026] Compared with the existing technical solutions, the above one or more technical solutions have the following beneficial effects:

[0027] This technical approach demonstrates, for the first time, abnormal lipid accumulation in CLL cells and overexpression of ACLY, a key enzyme in fatty acid synthesis, in CLL. ACLY serves as a hub for lipid, cholesterol, and acetylation in CLL. Furthermore, ACLY is regulated by the PI3K-AKT pathway and increases sensitivity to idelalisib. Therefore, ACLY may serve as a potential target for fatty acid metabolism intervention in the treatment of CLL.

[0028] In summary, the research results of the present invention indicate that ACLY acts as a hub connecting cell metabolic epigenetics in chronic lymphocytic leukemia, and reveals the molecular mechanism of chronic lymphocytic leukemia cell proliferation, which helps to improve the treatment effect and therefore has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1Figures 1 and 2 show lipid accumulation and ACLY overexpression in chronic lymphocytic leukemia (CLL) according to the present invention. (A) Oil Red O staining of neutral lipids in B lymphocytes from healthy donors, CLL cell lines, and CLL patients. (B) Overexpression of ACLY mRNA in CLL samples GSE50006 and GSE31048 is high. (C) Overexpression of ACLY mRNA in primary CLL cells is high. (D) In ​​CLL samples GSE22762, overexpression of ACLY is significantly associated with overall survival (OS). *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SEM.

[0031] Figure 2 ACLY regulates the proliferation, cell cycle, and apoptosis of chronic lymphocytic leukemia cells, as described in the examples of the present invention. (A) qRT-PCR was used to verify the efficiency of ACLY knockdown in chronic lymphocytic leukemia cells. (B) OD values ​​of chronic lymphocytic leukemia cells after ACLY knockdown using CCK-8. (C) Flow cytometry was used to assess apoptosis in ACLY knockdown cells. (D) ACLY knockdown regulates the expression of apoptosis-related proteins. (E) ACLY knockdown induces cell cycle arrest at the G0 / G1 phase and (F) inhibits the expression of CyclinD1 and CDK2. *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SEM.

[0032] Figure 3 BMS-303141 demonstrates anti-leukemic effects in chronic lymphocytic leukemia (CLL) through targeted inhibition of ACLY in CLL cells according to the present invention. (A) BMS-303141 inhibits the proliferation of CLL cell lines and primary CLL cells in a time- and dose-dependent manner. (B) BMS-303141 reduces ACLY protein expression in CLL cells in a dose-dependent manner. (C) Flow cytometry analysis of apoptosis and relative apoptosis rates in cells treated with BMS-303141. (D) BMS-303141 regulates the expression of apoptosis-related proteins. (E) and (F) are protein electrophoresis and flow cytometric images, respectively, of BMS-303141-induced cell cycle arrest at the G0 / G1 phase. (G) BMS-303141 inhibits CDK2 expression. *p<0.05, **p<0.01, ***p<0.001; mean ± SEM.

[0033] Figure 4ACLY promotes idelalisib sensitivity in the present invention. (A) MEC1, (B) EHEB, (C) cell survival after idelalisib treatment in patients with ACLY knockout in chronic lymphocytic leukemia. (D) MEC1, (E) EHEB, (F, G, H) cell survival after idelalisib and BMS-303141 combination therapy in different chronic lymphocytic leukemia patients. *p < 0.05, **p < 0.01, ***p < 0.001; mean ± SD. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0036] In view of this, a typical embodiment of the present invention provides a reagent for detecting the expression level of ATP-citrate lyase for use in the preparation of a chronic lymphocytic leukemia assessment product. Specifically, the present invention has found through research that compared with normal lymphocytes, the lipid levels of chronic lymphocytic leukemia cell lines and cells from chronic lymphocytic leukemia patients are significantly elevated, and ACLY is overexpressed in chronic lymphocytic leukemia patients. At the same time, chronic lymphocytic leukemia patients with higher ACLY expression have a shorter overall survival (OS), that is, ACLY overexpression is associated with lipogenesis and poor prognosis in chronic lymphocytic leukemia.

[0037] Therefore, the chronic lymphocytic leukemia assessment product specifically assesses the adipogenesis and / or prognosis of chronic lymphocytic leukemia.

[0038] The product may be a test kit, a test device and / or an apparatus.

[0039] In one or more specific embodiments of the present invention, a system for evaluating chronic lymphocytic leukemia is provided, the system comprising:

[0040] i) an analysis unit, the analysis unit comprising: a detection reagent for determining the expression level of the above-mentioned ATP-citrate lyase in a test sample of a subject, and;

[0041] ii) an evaluation unit, comprising: evaluating the disease condition of the subject according to the expression level of the ATP-citrate lyase determined in i).

[0042] The subject may be a human or a non-human mammal, with humans being preferred. The sample to be tested includes peripheral blood, and further includes peripheral blood mononuclear cells (PBMCs).

[0043] In one or more specific embodiments of the present invention, there is provided the use of ATP-citrate lyase as a target in the preparation and / or screening of chronic lymphocytic leukemia drugs.

[0044] The chronic lymphocytic leukemia drug includes drugs for preventing and / or treating chronic lymphocytic leukemia.

[0045] In one or more specific embodiments of the present invention, there is provided the use of an ATP-citrate lyase inhibitor in any one or more of the following:

[0046] (a) preparing products for inhibiting the proliferation of chronic lymphocytic leukemia cells and / or promoting apoptosis and cell cycle arrest of chronic lymphocytic leukemia cells;

[0047] (b) preparing a product for increasing the sensitivity of chronic lymphocytic leukemia to PI3K inhibitors;

[0048] (c) Preparation of anti-chronic lymphocytic leukemia products.

[0049] The ATP-citrate lyase inhibitors include, but are not limited to, RNA interference molecules or antisense oligonucleotides targeting the ATP-citrate lyase encoding gene, small molecule inhibitors, shRNA, siRNA, substances for implementing lentiviral infection or gene knockout (such as CRISPR / Cas9), and specific antibodies against ATP-citrate lyase itself or its upstream and downstream molecules, such as anti-ACLY antibodies, and may also include compound inhibitors. In one embodiment of the present invention, the ATP-citrate lyase inhibitor includes BMS-303141.

[0050] The product may be a drug or a test reagent, and the test reagent is used for basic research.

[0051] In one or more specific embodiments of the present invention, there is provided the use of an ATP-citrate lyase inhibitor combined with a PI3K inhibitor in the preparation of a drug for chronic lymphocytic leukemia.

[0052] The ATP-citrate lyase may be BMS-303141, the PI3K inhibitor may be idelalisib, and the molar ratio of BMS-303141 to idelalisib is 12:5-35.

[0053] In one or more specific embodiments of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition include at least an ATP-citrate lyase inhibitor and a PI3K inhibitor.

[0054] The active ingredients of the composition include at least BMS-303141 and idelalisib. The combination of the two can significantly inhibit the proliferation of chronic lymphocytic leukemia cells and reduce the survival rate of chronic lymphocytic leukemia cells, showing significant drug synergistic effect.

[0055] In one or more specific embodiments of the present invention, the molar ratio of BMS-303141 to idelalisib is 12:5-35.

[0056] The pharmaceutical composition may further include at least one pharmaceutically inactive ingredient.

[0057] The inactive pharmaceutical ingredients may be carriers, excipients, and diluents commonly used in pharmacy. Furthermore, according to conventional methods, the pharmaceutical composition may be prepared into oral preparations, external preparations, suppositories, and sterile injectable solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0058] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0059] In another embodiment of the present invention, the medicine of the present invention can be administered to the body in a known manner. For example, it can be delivered to the tissue of interest by systemic intravenous delivery or local injection. Alternatively, it can be administered intravenously, percutaneously, intranasally, through the mucosa, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on various factors to a great extent, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0060] In one or more specific embodiments of the present invention, a method for treating chronic lymphocytic leukemia is provided, comprising administering the ATP-citrate lyase inhibitor or the pharmaceutical composition to a patient.

[0061] The present invention is further described below with reference to specific examples. The following examples are intended only to illustrate the present invention and are not intended to limit its contents. If the specific experimental conditions are not specified in the examples, conventional conditions or conditions recommended by the sales company are generally followed; unless otherwise specified in the present invention, all can be purchased through commercial channels.

[0062] Example

[0063] Materials and Methods

[0064] clinical specimens

[0065] Samples from 87 newly diagnosed chronic lymphocytic leukemia patients with informed consent were collected from the hematology database of the Shandong Provincial Hospital for Chronic Lymphocytic Leukemia (SPHCLL). The diagnosis of chronic lymphocytic leukemia was based on the revised International Workshop on Chronic Lymphocytic Leukemia (IWCLL) criteria. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation as previously reported.

[0066] Cell lines and reagents

[0067] MEC-1 and EHEB cells were maintained in IMDM (MEC-1) and RPMI-1640 (EHEB and primary cells) culture medium supplemented with 10% heat-inactivated fetal bovine serum (Gibco, MD, USA), 1% penicillin / streptomycin mixture, and 2 mM l-glutamine. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. All cells were regularly checked for mycoplasma infection. For in vitro experiments, BMS-303141 (MCE, Shanghai, China) was used, soluble in DMSO (Solarbio, Beijing, China).

[0068] RNA isolation and real-time PCR

[0069] Total RNA was purified using Trizol reagent (Invitrogen, MA, USA) and reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qPCR) was performed using the Light Cycler 480 II real-time PCR (qPCR) system (Roche Diagnostics, Basel, Switzerland) with SYBR Green (AG, China). qPCR was performed in triplicate for each sample. Results were obtained using the Light Cycler 480 sequence detection software and analyzed using GraphPad Prism 7.0 statistical software.

[0070] Western blotting

[0071] Cell lysates were extracted with radioimmunoprecipitation buffer and phosphatase inhibitors (PhosSTOP, Roche, Basel, Switzerland). The following primary antibodies were used: anti-ACLY (ab40793, Abcam), anti-acetylated-lysine (sc32268, Santa Cruz), anti-CDK2 (18048, CST), anti-PARP (9532, CST), anti-Bcl2 (3498, CST), anti-BAX (14796, CST), anti-ABCA1 (ab307534, Abcam), and anti-GAPDH (TA-09, ZSGB).

[0072] Cytotoxicity assay

[0073] The viability of chronic lymphocytic leukemia cells was determined in triplicate and analyzed using a cell counting kit-8 (Dojindo Laboratories, Kumamoto, Japan). The cell density was simply adjusted to 1 × 10 4 Cells were seeded in 96-well plates at a concentration of 100 μl per well. CCK8 reagent was then added to each well and incubated for 3 hours. Finally, optical density was measured at 450 nm using a SpectraMax M2 microplate reader (Molecular Devices, CA, USA) to determine cell proliferation.

[0074] Cell cycle and apoptosis assays

[0075] Cell cycle analysis was performed using the propidium iodide cell cycle reagent protocol. Briefly, cells were harvested and fixed with 70% ethanol overnight at 4°C. After fixation, samples were stained with PI / RNase staining buffer (BD Biosciences) for 15 minutes. Apoptosis was detected using the Annexin V-PE / 7AAD Apoptosis Detection Kit according to the manufacturer's instructions (BD Biosciences). Cell cycle distribution analysis and apoptotic cell percentage were measured by flow cytometry using a Navios flow cytometer (Beckman Coulter, CA, USA).

[0076] Red Oil O Staining

[0077] Fresh chronic lymphocytic leukemia cells were mounted on slides and fixed with Red Oil O (ORO) fixative for 15 minutes. The slides were then removed and air-dried for 15 minutes. The researchers then immersed the slides in ORO staining solution and stained them for 15 minutes. The slides were then rinsed with 60% isopropyl alcohol for 20 seconds, rinsed with tap water, and gently washed with distilled water. At the end of the procedure, cell nuclei were counterstained with hematoxylin for 3 minutes.

[0078] BODIPY staining

[0079] Because oleic acid is a potent inducer of triglyceride synthesis and storage, incubating cells with 30 μM oleic acid and bovine serum albumin serves as a positive control for increased neutral lipid content. BSA, which lacks fatty acids, serves as a control. At specific time points, prepare a 2 μM BODIPY staining solution in PBS. Cells are then washed with 3 ml of PBS and incubated in 3 ml of staining solution at 37°C for 15 minutes. Wash twice with 3 ml of PBS and finally stain with DAPI. Slides should be imaged immediately under a microscope.

[0080] Immunofluorescence

[0081] MEC-1 and EHEB cells were pressed onto slides, incubated in methanol at -10°C for 5 minutes, and air-dried. Next, the slides were blocked with 5% goat serum for 1 hour and then incubated with antibodies overnight at 4°C. After a further incubation with Dylight 488-labeled goat anti-rat IgG antibody (Abbkine, Beijing, China) for 1 hour at room temperature, the slides were washed and stained with DAPI. Microanalysis was performed under a Nikon C2 confocal microscope.

[0082] Co-immunoprecipitation (Co-IP) assay

[0083] Cells were harvested and lysed with Co-IP lysis buffer. After centrifugation, 1–3 μg of primary antibody was added to the supernatant in lysis buffer, shaken, and incubated overnight at 4°C. The next day, Protein A / G PLUS agarose beads (Santa Cruz Biotechnology, USA) were added to the buffer to bind to the antibody, shaken, and incubated at 4°C for 2 h. The beads were washed three times with PBS and denatured by heating at 100°C. Proteins were then detected by western blotting.

[0084] Statistical analysis

[0085] All data were statistically analyzed using SPSS 26.0 (Chicago, IL, USA) and Graphpad Prism 8.0 (San Diego, CA, USA). The results of three independent experiments are presented as mean ± SEM. Direct comparisons were performed with the Student's t-test and Mann-Whitney U test. Multiple group comparisons were performed with one-way analysis of variance or two-way analysis of variance. Survival was analyzed with the Kaplan-Meier method. *P < 0.05 indicated statistical significance. The correlation between ACLY and ABCA1 expression was calculated using Spearman rank correlation.

[0086] result

[0087] ACLY overexpression is associated with adipogenesis and poor prognosis in chronic lymphocytic leukemia

[0088] Abnormal lipid accumulation has been found in many types of tumors. To investigate lipogenesis in CLL cells and the expression of the key enzyme ACLY, we analyzed the lipid content and ACLY mRNA levels in the cytoplasm of CLL cells. The results showed that compared with normal lymphocytes, the lipid levels in CLL cell lines and CLL patient cells were significantly increased ( Figure 1 A). Statistical analysis of ACLY expression based on GSE50006 and GSE31048 showed that ACLY was overexpressed in patients with chronic lymphocytic leukemia ( Figure 1 B). Subsequent qRT-PCR analysis of chronic lymphocytic leukemia specimens confirmed these results ( Figure 1 C). In addition, based on GSE22762 analysis, patients with chronic lymphocytic leukemia with higher ACLY expression had shorter overall survival (OS) ( Figure 1 D) This suggests that upregulation of ACLY may be involved in adipogenesis and progression of chronic lymphocytic leukemia.

[0089] ACLY knockout reduces chronic lymphocytic leukemia cell proliferation, enhances apoptosis, and is accompanied by cell cycle arrest

[0090] To evaluate the functional role of ACLY, we designed three small interfering RNAs (si-ACLY#1: CCGGCCUCUUCAAUUUCUATT, SEQ ID NO.1; UAGAAAUUGAAGAGGCCGGTT, SEQ ID NO.2; si-ACLY#2: GGCACAAAGAGAUCCUGAUTT, SEQ ID NO.3; AUCAGGAUCUCUUUGUGCCTT, SEQ ID NO.4; si-ACLY#3: GCACUGAGGAAUAUAAGAUTT, SEQ ID NO.5; AUCUUAUAUUCCUCAGUGCTT, SEQ ID NO.6) to reduce the expression of ACLY in chronic lymphocytic leukemia cell lines and verified the transfection efficiency by qRT-PCR. The latter two si-RNAs showed a significant decrease in ACLY mRNA levels (p < 0.05) ( Figure 2 A). CCK-8 assay showed that ACLY knockdown significantly reduced the proliferation capacity of chronic lymphocytic leukemia cells ( Figure 2 B). Subsequently, flow cytometry showed that ACLY knockdown increased the rate of cell apoptosis ( Figure 2C). In addition, after knockdown of ACLY, the expression of pro-apoptotic proteins Bax and PARP increased, and the expression of anti-apoptotic protein Bcl-2 decreased ( Figure 2 D). We further analyzed the regulatory effect of ACLY on the cell cycle and found that the proportion of cells in the G0 / G1 phase increased significantly (p<0.05) ( Figure 2 E). In addition, ACLY knockdown down-regulated the expression of Cyclin D1 and CDK2, inhibiting the transition of the cell cycle from G1 phase to S phase ( Figure 2 F). In conclusion, our results suggest that ACLY promotes the progression of chronic lymphocytic leukemia by regulating proliferation, cell cycle, and apoptosis.

[0091] BMS-303141 targeted inhibition of ACLY has anti-leukemic effects in chronic lymphocytic leukemia

[0092] We next evaluated the efficacy of the ACLY inhibitor BMS-303141 in the treatment of chronic lymphocytic leukemia. Chronic lymphocytic leukemia cells and primary cells from chronic lymphocytic leukemia patients were incubated with BMS-303141 at the indicated concentrations for 24 to 72 hours. Cell proliferation was dose-dependent and time-dependent ( Figure 3 A). In addition, according to Western blotting, ACLY was decreased in a dose-dependent manner in chronic lymphocytic leukemia cells treated with BMS-303141 ( Figure 3 B). Next, flow cytometry showed that the apoptosis rate of chronic lymphocytic leukemia cells cultured with BMS-303141 increased in a dose-dependent manner ( Figure 3 C, D). It is noteworthy that with the increase of BMS-303141 concentration, the expression of apoptotic proteins (Bax and cleaved-PARP) was upregulated ( Figure 3 E). In addition, BMS-303141 induced an increase in G0 / G1 phase cells in a dose-dependent manner ( Figure 3 F). After BMS-303141 treatment, the expression of CDK2 also decreased in a dose-dependent manner ( Figure 3 G) ACLY is regulated by the PI3K-AKT pathway and increases idelalisib sensitivity.

[0093] Previous studies have shown that the activity of ACLY is regulated by phosphorylation. WB showed that after treatment with Idelalisib in chronic lymphocytic leukemia cell lines MEC1, EHEB and primary cells of chronic lymphocytic leukemia patients, the protein levels of PI3K, p-AKT, and p-ACLY decreased with increasing drug concentration. There was no obvious change in the protein levels of AKT and ACLY. This shows that in chronic lymphocytic leukemia, the phosphorylation level of ACLY is regulated by the PI3K-AKT pathway. Therefore, we speculate that knocking down ACLY can increase the sensitivity of the PI3K inhibitor Idelalisib. Cytotoxicity experiments showed that after knocking out ACLY in chronic lymphocytic leukemia cell lines and primary cells, the sensitivity of the Idelalisib drug increased and the cell survival rate was significantly reduced ( Figure 4 AC). At the same time, the combined use of Idelalisib and BMS-303141 significantly reduced the survival rate of chronic lymphocytic leukemia cells ( Figure 4 DH), the CI values ​​of the combined use of the drugs are shown in Tables 1-5, indicating that the combination of the two produces a synergistic anti-tumor effect.

[0094] Table 1 CI values ​​of Idelalisib and BMS-303141 combined in MEC1 cells

[0095] Idelalisib dose (μM) BMS-303141 dose (μM) Effect of combined medication CI 25 60 0.40326 0.38245 50 60 0.42151 0.69757 75 60 0.53018 0.61783 100 60 0.58813 0.62065 125 60 0.67712 0.48868 150 60 0.74630 0.39038 175 60 0.82930 0.24914

[0096] Table 2 CI values ​​of Idelalisib and BMS-303141 combined in EHEB cells

[0097]

[0098]

[0099] Table 3 CI values ​​of idelalisib and BMS-303141 combined in 98 CLL patients

[0100] Idelalisib dose (μM) BMS-303141 dose (μM) Effect of combined medication CI 25 60 0.27747 0.16132 50 60 0.29483 0.27040 75 60 0.30755 0.36253 100 60 0.35333 0.33781 125 60 0.36521 0.38619 150 60 0.37772 0.42265 175 60 0.40391 0.40876

[0101] Table 4 CI values ​​after combination of Idelalisib and BMS-303141 in CLL patient 128

[0102] Idelalisib dose (μM) BMS-303141 dose (μM) Effect of combined medication CI 25 60 0.57227 0.06368 50 60 0.59422 0.10470 75 60 0.60364 0.14426 100 60 0.62671 0.15582 125 60 0.63502 0.18031 150 60 0.63618 0.21405 175 60 0.65564 0.20777

[0103] Table 5 CI values ​​after combination of Idelalisib and BMS-303141 in CLL patient 143

[0104] Idelalisib dose (μM) BMS-303141 dose (μM) Effect of combined medication CI 25 60 0.52055 0.19693 50 60 0.52662 0.38246 75 60 0.60660 0.38913 100 60 0.60613 0.52003 125 60 0.65581 0.50430 150 60 0.65166 0.61847 175 60 0.66182 0.68392

[0105] In summary, the present study observed abnormal lipid accumulation in CLL cells and overexpression of ACLY, a key enzyme in fatty acid synthesis, in CLL. ACLY serves as a hub for lipid, cholesterol, and acetylation in CLL. Furthermore, ACLY is regulated by the PI3K-AKT pathway and increases sensitivity to idelalisib. Therefore, ACLY may serve as a potential target for fatty acid metabolism intervention in the treatment of CLL.

[0106] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of ATP-citrate lyase inhibitors combined with PI3K inhibitors in the preparation of drugs for chronic lymphocytic leukemia; The ATP-citrate lyase inhibitor is BMS-303141, and the PI3K inhibitor is idelalisib; The BMS-303141 dose was 60 µM; the idelalisib dose was 25 µM.

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