Application of KRAS to regulation of fatty acid metabolism in chronic lymphocytic leukemia

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of KRAS to regulation of fatty acid metabolism in chronic lymphocytic leukemia. Specifically, the regulation and control effect and mechanism of KRASG13D mutation on CLL fatty acid oxidation and malignant progression are disclosed for the first time through research, the relationship between KRAS mutation and CLL progression is disclosed from the perspective of energy metabolism, and the KRASG13D mutation activates a downstream PI3K / AKT / mTOR signal channel through up-regulation of PIK3C2A, so that CLL cell fatty acid oxidation and proliferation activity enhancement are promoted. The PI3K inhibitor is applied to treatment of recurrent and refractory CLL, however, the research finds that KRASG13D mutant CLL cells show the drug resistance tendency of the PI3K inhibitor, and the treatment effect of the PI3K inhibitor can be improved by combined application of the KRAS inhibitor, so that a clue is provided for further knowing the occurrence and development mechanism of CLL and developing a new strategy for clinical targeted treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and molecular biology, and specifically relates to the application of KRAS in regulating fatty acid metabolism in chronic lymphocytic leukemia. Background Art

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

[0003] Chronic lymphocytic leukemia (CLL) is a hematological malignancy characterized by clonal proliferation of mature B lymphocytes. Its signature immune markers include CD19, CD5, CD20, CD23, κ, and λ. In recent years, significant progress has been made in the treatment of CLL. Targeted drugs represented by Bruton's Tyrosine Kinase (BTK) inhibitors, BCL-2 inhibitors, and PI3K inhibitors have significantly improved the prognosis of patients. However, drug-resistant relapse and immune microenvironment-mediated treatment resistance are still clinical problems that need to be solved urgently. It is crucial to deeply analyze the regulatory mechanism of CLL malignant progression.

[0004] The molecular genetic heterogeneity of CLL is the core driving factor for the differences in its clinical outcomes. Common molecular genetic abnormalities include chromosomal abnormalities such as del(17p), del(11q), del(13q), +12, and gene mutations such as TP53, NOTCH1, and SF3B1. However, the genetic landscape of CLL still cannot fully explain its heterogeneous progression. In recent years, the pivotal role of metabolic reprogramming has gradually attracted attention. Unlike the abnormal glucose metabolism dominated by the Warburg effect in solid tumors, lipid metabolic reprogramming regulates the occurrence and development of CLL in multiple dimensions. On the one hand, CLL cell survival and proliferation are highly dependent on fatty acid oxidation (FAO) for energy supply. On the other hand, signaling molecules such as prostaglandins derived from lipid peroxidation can inhibit T cell anti-tumor immunity and induce immune escape microenvironment. Revealing the genetic basis of CLL metabolic reprogramming has become the key to breaking through the existing treatment bottleneck.

[0005] The oncogene KRAS is a core member of the RAS / MAPK signaling pathway, and its mutation has been widely confirmed in solid tumors to regulate the metabolic phenotype of cancer. However, the metabolic regulatory function of KRAS mutation in hematological tumors, especially CLL, is still rarely studied. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide the application of KRAS in regulating fatty acid metabolism in chronic lymphocytic leukemia. Specifically, the present invention for the first time reveals through research that KRAS G13D mutation regulates the fatty acid oxidation and malignant progression of CLL and its mechanism, reveals the relationship between KRAS mutation and CLL progression from the perspective of energy metabolism, and proposes that KRAS G13D mutation promotes the fatty acid oxidation and proliferation activity enhancement of CLL cells by upregulating PIK3C2A to activate the downstream PI3K / AKT / mTOR signaling pathway. PI3K inhibitors have been applied to the treatment of relapsed and refractory CLL. However, this study found that KRAS G13D mutant CLL cells showed a tendency to be resistant to PI3K inhibitors, and the combined application of KRAS inhibitors could improve the therapeutic effect of PI3K inhibitors. Based on the above research results, the present invention is completed.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided the application of a reagent for detecting KRAS mutation in any one or more of the following:

[0009] (a1) Preparing a product for prognostic assessment of chronic lymphocytic leukemia;

[0010] (a2) Preparing a product for detecting drug sensitivity of chronic lymphocytic leukemia.

[0011] Specifically, the KRAS mutation is KRAS G13D , and the present invention discovers through experiments that KRAS G13D mutation is a hot mutation of KRAS in CLL patients, which can promote the proliferation of CLL cells, enhance the fatty acid oxidation activity, is significantly associated with poor prognosis of CLL patients, and at the same time induces a decrease in the sensitivity of CLL cells to PI3K inhibitors.

[0012] In (a2), the drug for chronic lymphocytic leukemia can be a PI3K inhibitor; more specifically, the PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335.

[0013] In the second aspect of the present invention, there is provided the application of a KRAS mutation or a reagent promoting KRAS mutation in at least one of the following (b1)-(b5):

[0014] (b1) Upregulating PIK3C2A to regulate the PI3K / AKT / mTOR signaling pathway or preparing a product for upregulating PIK3C2A to regulate the PI3K / AKT / mTOR signaling pathway;

[0015] (b2)Enhancing fatty acid oxidation in CLL cells or preparing a product for enhancing fatty acid oxidation in CLL cells;

[0016] (b3)Promoting the proliferation of CLL cells or preparing a product for promoting the proliferation of CLL cells;

[0017] (b4)Enhancing the resistance of CLL cells to PI3K inhibitors or preparing a product for enhancing the resistance of CLL cells to PI3K inhibitors;

[0018] (b5)Constructing a CLL-related disease model.

[0019] The product can be a drug or an experimental reagent for non-medical use, so as to be available for basic research.

[0020] The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335.

[0021] The construction of the CLL-related disease model includes constructing a CLL cell disease model or a CLL animal disease model.

[0022] In a third aspect of the present invention, there is provided the use of a reagent for inhibiting KRAS mutations in at least one of the following (c1)-(c5):

[0023] (c1)Inhibiting the upregulation of the PIK3C2A-regulated PI3K / AKT / mTOR signaling pathway or preparing a product for inhibiting the upregulation of the PIK3C2A-regulated PI3K / AKT / mTOR signaling pathway;

[0024] (c2)Inhibiting fatty acid oxidation in CLL cells or preparing a product for inhibiting fatty acid oxidation in CLL cells;

[0025] (c3)Inhibiting the proliferation of CLL cells or preparing a product for inhibiting the proliferation of CLL cells;

[0026] (c4)Inhibiting the resistance of CLL cells to PI3K inhibitors or preparing a product for inhibiting the resistance of CLL cells to PI3K inhibitors;

[0027] (c5)A product for treating CLL.

[0028] The CLL can be KRAS mutant CLL, further including KRAS G13D mutant CLL.

[0029] The product can be a drug or an experimental reagent for non-medical use, so as to be available for basic research.

[0030] According to the present invention, when the product is a drug, the drug further includes at least one pharmaceutically inactive ingredient.

[0031] The pharmaceutical inactive ingredients may be carriers, excipients, diluents, etc. commonly used in pharmacy. Moreover, according to common methods, it can be made into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injection solutions for use.

[0032] The non-drug active ingredients such as carriers, excipients, and diluents that may be included are well-known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

[0033] Preferably, the carriers, excipients, and diluents include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, etc.

[0034] Preferably, the drug of the present invention can be administered into the body by known means. For example, it can be delivered systemically via intravenous injection or locally injected into the tissue of interest. Optionally, it can be administered via intravenous, transdermal, intranasal, mucosal, or other delivery methods. Such administration can be carried out via a single dose or multiple doses. Those skilled in the art understand that the actual dose to be administered in the present invention can vary to a large extent depending on various factors, such as target cells, biological type or its tissue, the general condition of the subject to be treated, the administration route, the administration method, and so on.

[0035] Preferably, the drug administration objects can be humans and non-human mammals, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, chimpanzees, etc.

[0036] The reagent for inhibiting KRAS mutation includes a KRAS inhibitor, specifically BI-2865 (Cas No.: 2937327-93-8).

[0037] The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335 (Cas No.: 2569296-51-9).

[0038] In the fourth aspect of the present invention, there is provided the use of a KRAS inhibitor in combination with a PI3K inhibitor in the preparation of a therapeutic drug for CLL.

[0039] Among them, the KRAS inhibitor, specifically, can be BI-2865 (Cas No.: 2937327-93-8).

[0040] The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335 (Cas No.: 2569296-51-9).

[0041] The molar ratio of the KRAS inhibitor to the PI3K inhibitor is 1:0.01 - 10.

[0042] The CLL can be KRAS - mutant CLL, further including KRAS G13D mutant CLL.

[0043] In a fifth aspect of the present invention, there is provided a composition, the active ingredients of which at least comprise a KRAS inhibitor and a PI3K inhibitor.

[0044] Among them, the KRAS inhibitor can specifically be BI - 2865 (Cas No.: 2937327 - 93 - 8).

[0045] The PI3K inhibitor is the PIK3C2A inhibitor MIPS - 21335 (Cas No.: 2569296 - 51 - 9).

[0046] The molar ratio of the KRAS inhibitor to the PI3K inhibitor is 1:0.01 - 10.

[0047] The composition is used for treating CLL, especially KRAS - mutant CLL (including KRAS G13D mutant CLL), indicating that dual inhibition targeting KRAS and PI3K may exert a stronger anti - tumor effect by synergistically blocking signal crosstalk.

[0048] In a sixth aspect of the present invention, there is provided a method for treating CLL, the method comprising administering the above - mentioned composition to a subject. Among them, the CLL includes KRAS - mutant CLL (further including KRAS G13D mutant CLL).

[0049] The beneficial technical effects of the above - mentioned one or more technical solutions:

[0050] The above - mentioned technical solutions have systematically elucidated for the first time the biological significance of KRAS G13D mutation in CLL and its molecular mechanism promoting disease progression. The KRAS G13D mutation is a hot - spot mutation of the KRAS gene in CLL patients. Although its mutation frequency in CLL is low, it is significantly associated with poor prognosis of patients. This study found that KRAS G13D mutation significantly promotes the proliferation and fatty acid oxidation activity of CLL cells. At the molecular mechanism level, we found that KRAS G13D mutation activates the PI3K / AKT / mTOR signaling pathway by up - regulating the expression of PIK3C2A, thereby enhancing fatty acid oxidation and promoting the proliferation of CLL cells. In addition, KRAS G13DThe mutation also significantly inhibits the sensitivity of CLL cells to PI3K inhibitor drugs. When the KRAS inhibitor BI-2865 and the PIK3C2A inhibitor MIPS-21335 are used in combination, the activity of KRAS G13D CLL cells is significantly reduced, suggesting that dual inhibition targeting KRAS and PI3K may exert a stronger anti-tumor effect by synergistically blocking signal crosstalk. These findings start from fatty acid metabolism, reveal the relationship between KRAS mutation and CLL progression, and provide clues for further understanding the mechanism of CLL occurrence and development and developing new clinical targeted treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0052] Figure 1 For the KRAS in the embodiments of the present invention G13D mutation promotes the proliferation of CLL cells.

[0053] Figure 2 For the KRAS inhibitor in the embodiments of the present invention to inhibit KRAS G13D mutation in CLL cells from proliferating.

[0054] (A) The cell proliferation activity of MEC1 cells transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with the KRAS inhibitor BI-2865 for 24 hours. The drug concentration gradients are 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM. (B) The cell proliferation activity of EHEB cells transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with the KRAS inhibitor BI-2865 for 24 hours. (C) The cell proliferation activity of Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with the KRAS inhibitor BI-2865 for 24 hours. (D) The cell proliferation activity of Pt#2 primary cells transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with the KRAS inhibitor BI-2865 for 24 hours.

[0055] Figure 3 For the KRAS mutation in the embodiments of the present invention to regulate the transcriptional levels of key enzymes in fatty acid oxidation of CLL cell lines.

[0056] (A-E) Cells transfected with KRAS WT and KRAS G13DTranscription levels of key fatty acid oxidation enzymes CPT1A, CPT2, ACLS1, ACOX1, and HADHA in MEC1 cells with mutant plasmids. (F-J) Transfection of KRAS WT and KRAS G13D Transcription levels of key fatty acid oxidation enzymes CPT1A, CPT2, ACLS1, ACOX1, and HADHA in EHEB cells with mutant plasmids

[0057] Figure 4 This is the regulation of the transcription levels of key fatty acid oxidation enzymes in primary CLL cells by KRAS mutation in the examples of the present invention

[0058] (A-E) Transcription levels of key fatty acid oxidation enzymes CPT1A, CPT2, ACLS1, ACOX1, and HADHA in Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids. (F-J) Transfection of KRAS WT and KRAS G13D Transcription levels of key fatty acid oxidation enzymes CPT1A, CPT2, ACLS1, ACOX1, and HADHA in Pt#2 primary cells transfected with mutant plasmids. (K-O) Transfection of KRAS WT and KRAS G13D Transcription levels of key fatty acid oxidation enzymes CPT1A, CPT2, ACLS1, ACOX1, and HADHA in Pt#3 primary cells transfected with mutant plasmids

[0059] Figure 5 This is the enhancement of oxygen consumption in CLL cells by KRAS mutation in the examples of the present invention

[0060] (A) Extracellular oxygen consumption levels of MEC1 cells transfected with KRAS WT and KRAS G13D mutant plasmids. Left: Line graph of real-time fluorescence detection of extracellular oxygen consumption; Right: Comparison of extracellular oxygen consumption of KRAS WT and KRAS G13D (B) Extracellular oxygen consumption levels of Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids. (C) Extracellular oxygen consumption levels of Pt#2 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids. (D) Extracellular oxygen consumption levels of Pt#4 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids

[0061] Figure 6 This is the enhancement of fatty acid oxidation levels in CLL cells by KRAS mutation in the examples of the present invention

[0062] (A) Fatty acid oxidation level of Pt#2 primary cells transfected with KRAS WT plasmid. (B) Fatty acid oxidation level of Pt#2 primary cells transfected with KRAS G13D plasmid. (C) Comparison of fatty acid oxidation levels in KRAS WT and KRAS G13D Pt#2 primary cells. (D) Fatty acid oxidation level of Pt#3 primary cells transfected with KRAS WT plasmid. (E) Fatty acid oxidation level of Pt#3 primary cells transfected with KRAS G13D plasmid. (F) Comparison of fatty acid oxidation levels in KRAS WT and KRAS G13D Pt#3 primary cells.

[0063] Figure 7 For the KRAS in the embodiments of the present invention G13D and KRAS WT CLL cell transcriptome analysis.

[0064] (A) Volcano plot of differential expression analysis of the transcriptome of KRAS WT and KRAS G13D mutant MEC1 cells. (B) ssGSEA analysis results of fatty acid metabolism-related pathways in KRAS WT and KRAS G13D mutant MEC1 cells. (C) GO enrichment analysis results of KRAS WT and KRAS G13D mutant MEC1 cells.

[0065] Figure 8 For the KRAS in the embodiments of the present invention G13D activates the expression of PIK3C2A in CLL cells.

[0066] (A) Transcription level of PIK3C2A in MEC1 cells transfected with KRAS WT and KRAS G13D mutant plasmids. (B) Transcription level of PIK3C2A in EHEB cells transfected with KRAS WT and KRAS G13D mutant plasmids. (C) Transcription level of PIK3C2A in Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids. (D) Transcription level of PIK3C2A in Pt#2 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids.

[0067] Figure 9 In the embodiments of the present invention, KRAS G13D activates the PI3K / AKT / mTOR axis in CLL cells

[0068] Left: PI3K-2α, AKT, phosphorylated AKT, mTOR, and phosphorylated mTOR protein expression in MEC1 cells transfected with KRAS WT and KRAS G13D mutation plasmids. Right: PI3K-2α, AKT, phosphorylated AKT, mTOR, and phosphorylated mTOR protein expression in Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutation plasmids.

[0069] Figure 10 In the embodiments of the present invention, PI3K-C2α has multiple KRAS G13D binding sites.

[0070] (A-F) Visualization models of the KRASG13D and PI3K-C2α protein complex, showing 6 docking results with the lowest binding energy and the highest existence probability.

[0071] Figure 11 In the embodiments of the present invention, there is a protein interaction between KRAS and PI3K-2α.

[0072] The Co-IP experiment confirmed the protein interaction between KRAS and PI3K-2α. Left: PI3K-C2α was significantly enriched in the anti-KRAS immunoprecipitation complex; Left: KRAS was significantly enriched in the anti-PI3K-C2α immunoprecipitation complex.

[0073] Figure 12 In the embodiments of the present invention, KRAS G13D mutation reduces the sensitivity of CLL cells to PI3K inhibitors.

[0074] (A-C) Cell viability of MEC1 cells (A), EHEB cells (B), and Pt#2 primary cells (C) transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with idelalisib for 24 hours. The drug concentration gradient was 0 μM, 25 μM, 50 μM, 100 μM, 200 μM. (E-G) Cell viability of MEC1 cells (E), EHEB cells (F), and Pt#1 primary cells (G) transfected with KRAS WT and KRAS G13D mutation plasmids after treatment with MIPS-21335 for 24 hours. The drug concentration gradient was 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM.

[0075] Figure 13 In the embodiment of the present invention, the KRAS inhibitor and the PI3K inhibitor synergistically inhibit the proliferation of CLL cells.

[0076] (A) Cell viability of MEC1 cells transfected with KRAS WT and KRAS G13D mutant plasmids after co-treatment with MIPS-21335 and BI2865 for 24 hours. (B) Cell viability of Pt#1 primary cells transfected with KRAS WT and KRAS G13D mutant plasmids after co-treatment with MIPS-21335 and BI2865 for 24 hours. The drug concentration of MIPS-21335 was 1 μM, and the drug concentration gradient of BI2865 was 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM.

[0077] Figure 14 This is the result of the synergistic effect of the KRAS inhibitor and the PI3K inhibitor in the embodiment of the present invention.

[0078] (A) Dose-response heatmap of the combined application of BI2865 and MIPS-21335 at 0, 0.01, 0.1, 1, 10 μM respectively in KRAS G13D mutant Pt#1 primary cells. (B) Synergy score heatmap of the combined application of BI2865 and MIPS-21335 at 0, 0.01, 0.1, 1, 10 μM respectively in KRAS G13D mutant Pt#1 primary cells. (C) Dose-response heatmap of the combined application of BI2865 and MIPS-21335 at 0, 0.01, 0.1, 1, 10 μM respectively in KRAS G13D mutant MEC1 cells. (D) Synergy score heatmap of the combined application of BI2865 and MIPS-21335 at 0, 0.01, 0.1, 1, 10 μM respectively in KRAS G13D mutant MEC1 cells. Detailed implementation

[0079] It should be noted that the following detailed description is illustrative and is 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 of ordinary skill in the technical field to which the present application belongs.

[0080] 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 forms are also intended to include the plural forms. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0081] The present invention will be further described in conjunction with specific examples below. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0082] The present invention will be further explained and illustrated by the following examples, but it does not constitute a limitation to the present invention. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The test methods without specific conditions specified in the following examples are usually carried out under conventional conditions.

[0083] Example

[0084] 1. Materials and Methods

[0085] 1.1 Analysis of the Mutation Frequency of KRAS Gene in Patients with Chronic Lymphocytic Leukemia

[0086] The mutation information of a total of 3,205 patients with chronic lymphocytic leukemia from 9 independent cohorts was collected for statistical analysis (excluding cases with unclear KRAS gene mutation status). The mutation information in the cohorts included whole exome sequencing data and next-generation sequencing data. The number of cases with KRAS gene mutation was statistically analyzed and integrated, and the overall proportion of patients with chronic lymphocytic leukemia carrying the KRAS gene mutation was calculated, and the frequency of the measured KRAS gene mutation sites was analyzed.

[0087] Table 1 Information of Cohorts of Patients with Chronic Lymphocytic Leukemia

[0088]

[0089]

[0090] 1.2 Transcriptome Sequencing and Bioinformatics Analysis

[0091] The wild-type KRAS and KRAS G13D mutant MEC1 cell lines were respectively subjected to Illumina transcriptome sequencing to obtain the RNA expression profiles of the two groups of cells. FastQC was used to evaluate the quality of the original sequencing data (FASTQ files), and Trimmomatic was used to remove low-quality sequences (Phred score < 20), adapters, and reads with a length < 50 bp. Using the human genome GRCh38 as the reference genome, the gene-level counts matrix was calculated by featureCounts. DESeq2 was used to normalize the original counts data (based on the median ratio method), and RNA molecules differentially expressed between the two groups were identified with |log2fold change| > 1.2 and p < 0.05 as the statistical thresholds. clusterProfiler was used to further perform gene function (Gene Ontology, GO) enrichment analysis and single-sample gene set enrichment analysis (Single-sample gene set enrichment analysis, ssGSEA) on the transcriptome data.

[0092] 1.3 Immunoprecipitation experiment

[0093] (1) Collect 1×10 7 cells, centrifuge at 4°C and 500×g for 10 min, and discard the supernatant;

[0094] (2) Wash the cells twice with pre-cooled PBS, and add 200 μL of lysis buffer per 1×10 6 cells, and lyse on ice for 10 min;

[0095] (3) Centrifuge at 4°C and 14000×g for 10 min, and collect the supernatant for later use.

[0096] (4) Take 50 μL of magnetic bead suspension, add 400 μL of PBST buffer, and vortex to mix evenly;

[0097] (5) Place it on a magnetic stand for 1 min to separate, discard the supernatant, and repeat the washing 2 times.

[0098] (6) Antibody dilution: Dilute the primary antibody with PBST to a final concentration of 10 μg / mL;

[0099] (7) Incubation and binding: Add the diluted antibody to the washed magnetic beads, and mix by inversion at room temperature for 30 min;

[0100] (8) Washing: Wash the magnetic beads 4 times with PBST, 400 μL each time, and separate magnetically to discard the supernatant.

[0101] (9) Add 400 μL of antigen sample, and mix by inversion at room temperature for 30 min (or incubate at 4°C for 2 h);

[0102] (10) Wash 4 times with PBST, 400 μL each time, to completely remove non-specific binding.

[0103] (11) Add 50 μL of 1× SDS loading buffer to the magnetic beads, heat at 95 °C for 5 min, separate magnetically and collect the supernatant for subsequent Western Blot detection.

[0104] 1.4 Cell proliferation assay

[0105] (1) Collect sufficient amounts of experimental group and control group cells during the logarithmic growth phase of the cells. According to the cell density of 1×10 4 cells / 100 μl / well, seed the cells in a 96-well cell culture plate.

[0106] (2) Set 3 duplicate wells for both the experimental group and the control group cells, and set a blank control group.

[0107] (3) Incubate in a 37 °C incubator until the predetermined time point. When detecting, add 10 μl of CCK-8 reagent to the experimental group, control group, and blank control wells respectively, and incubate in the cell culture incubator for 1 - 4 h.

[0108] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 450 nm, and calculate the cell proliferation rate or survival rate.

[0109] 1.5 Detection experiment of extracellular oxygen consumption and fatty acid oxidation level

[0110] A. Cell pretreatment

[0111] (1) Collect sufficient amounts of experimental group and control group cells in the logarithmic growth phase, adjust the CLL cell density to 2×10 5 cells / ml, seed in a 96-well cell culture plate (100 μl per well), and set 6 replicates for each group.

[0112] (2) Set the experimental groups according to the kit instructions: basal FAO group (containing only oleic acid), Etomoxir inhibition group (oleic acid + 40 μM Etomoxir), FCCP activation group (oleic acid + 1 μM FCCP).

[0113] (3) Add 10 μl of oleic acid solution (final concentration 200 μM) to each well, and incubate at 37 °C for 30 min. Add Etomoxir or FCCP according to the groups, and add an equal volume of PBS to the control group.

[0114] B. Detection by machine

[0115] (1) Prepare the Extracellular O2 Consumption Reagent according to the kit instructions, add 20 μl to each well, and incubate in the dark for 10 min.

[0116] (2) Set the parameters of the microplate reader: excitation wavelength 380 nm, emission wavelength 650 nm, read the signal every 5 min for 2 h. Record the change in fluorescence signal related to oxygen consumption in real time (the signal intensity is negatively correlated with the extracellular oxygen concentration).

[0117] (3) Subtract the background signal (the average fluorescence value of the cell-free wells), and calculate the change rate of fluorescence intensity (ΔF / F0) at each time point. Basal fatty acid oxidation activity = the ΔF difference between the Etomoxir group and the basal group; maximum fatty acid oxidation activity = the ΔF difference between the FCCP group and the Etomoxir group.

[0118] 1.6 Virtual molecular docking

[0119] (1) The three-dimensional protein structures of KRAS G13D (PDB Entry ID: 8BLR) and PI3K-2α (PDB Entry ID: 7BI4) were obtained from the RCSB PDB database (https: / / www.rcsb.org). The three-dimensional protein structures should meet the following requirements: ① Resolution ≤ ② No significant missing residues or atoms; ③ Remove water molecules, cofactors, and irrelevant ligands.

[0120] (2) Use the GRAMM Web server for virtual protein docking. Set the docking mode to FreeDocking. Upload the three-dimensional protein structures in PDB format to the GRAMM server with the KRAS G13D protein as the ligand and the PI3K-2α protein as the receptor to predict the intermolecular interactions of the proteins and visualize the docking model.

[0121] (3) Use PyMOL 2.5.2 to load the docking results, and screen the conformation with the lowest binding free energy (ΔG) as the optimal model. Combine the known mutation experimental data or the binding sites reported in the literature to verify the biological rationality of the prediction results.

[0122] 1.7 Statistical analysis methods

[0123] Analysis of inter-group differences was performed using Student’s t-test (for two-sample comparison) or one-way / two-way ANOVA (for multi-group comparison). The raw data was sorted and preliminarily screened using Excel 2021, and outliers were identified and removed using Grubbs test. R 4.3.1, GraphPad Prism 9.0, and SPSS 26.0 were used for data visualization and basic statistical analysis. The experimental data was expressed as mean ± standard error (mean ± SEM). The significance levels were marked as: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0124] 2. Results

[0125] 2.1 KRAS G13D is a high-frequency mutation site in patients with chronic lymphocytic leukemia

[0126] To clarify the mutation characteristics of the KRAS gene in chronic lymphocytic leukemia, a total of 3,205 CLL patients who underwent whole-genome sequencing or next-generation sequencing were included in this study. Analysis of the KRAS mutation spectrum in the patients found that the overall mutation frequency of the KRAS gene in CLL was low, and CLL patients carrying KRAS mutations accounted for only 3.71% (119 / 3,205) of all the patients included in the study, but there was significant site heterogeneity. As shown in Table 2, among the 127 detected KRAS mutation sites, the G13D (p.Gly13Asp) site mutation had the highest proportion (29.13%, 37 / 127), which was significantly higher than other hot-spot mutations such as G12D (16.54%) or Q61H (0.79%). The analysis results showed that KRAS G13D mutations showed specific high-frequency mutations in CLL patients. Based on this, it was speculated that KRAS G13D had the potential to be a specific therapeutic target for CLL. Therefore, this study will mainly explore the impact of KRAS G13D mutations on the occurrence and development of CLL.

[0127] Table 2 KRAS mutation characteristics in CLL patients

[0128]

[0129]

[0130] 2.2 KRAS G13D mutations promote the proliferation of chronic lymphocytic leukemia cells

[0131] To clarify the involvement of KRAS mutations in the occurrence and development of CLL, KRAS WT (KRAS wildt ype, wild-type KRAS) and KRASG13D Mutant plasmids were transfected into CLL cell lines MEC1, EHEB and primary CLL cells. The cell proliferation activities were detected by CCK-8 assay at 24 hours, 48 hours and 72 hours respectively. The detection results showed that KRAS G13D mutation significantly promoted the proliferation of CLL cells. Compared with the KRAS WT group, the proliferation activities of MEC1 cells transfected with KRAS G13D ( Figure 1 A) increased by 1.3-fold, 1.3-fold and 1.1-fold at 24h, 48h and 72h respectively (p < 0.05); the proliferation activities of EHEB cells( Figure 1 B) showed a similar trend at the same time points (24h: 1.5-fold; 48h: 1.4-fold; 72h: 1.2-fold, p < 0.05). In primary CLL cells, compared with KRAS WT cells, the proliferation activity of KRAS G13D mutant cells also showed an increasing trend and was significantly higher than that of KRAS WT CLL cells at multiple time points. The difference in the proliferation activities of the two groups of Pt#3 primary cells persisted until 72h( Figure 1 C-E).

[0132] BI-2865 is a recently published non-covalent pan-KRAS inhibitor that effectively inhibits the activation of wild-type KRAS and various KRAS mutants by blocking nucleotide exchange, with an average IC50 of approximately 140 nM and dissociation constants of only 4.5 nM and 4.3 nM for G12D and G13D mutants, respectively, showing extremely high affinity. To further verify the effect of KRAS mutation on the proliferation of CLL cells, CLL cells were treated with the KRAS inhibitor BI-2865 at drug concentration gradients of 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM, and then the 24-hour proliferation activities of KRAS WT , KRAS G13D CLL cells were detected by CCK-8 assay respectively. The analysis results found that BI-2865 inhibited the proliferation of CLL cells, and the inhibitory effect was concentration-dependent, among which the inhibitory effect on the proliferation of CLL cells with KRAS G13D mutation was more significant( Figure 2 ). The above results indicate that KRAS G13D mutation can promote the proliferation of CLL cells, suggesting its potential driving role in disease progression.

[0133] 2.3 KRAS G13D mutation enhances the fatty acid oxidation level of chronic lymphocytic leukemia cells

[0134] As mentioned above, KRAS regulates fatty acid oxidation in a variety of solid tumors, and the survival and proliferation of CLL cells highly depend on energy supply from fatty acid oxidation. To explore whether there is fatty acid metabolic reprogramming in the regulation of CLL cell proliferation activity by KRAS G13D mutation, plasmids of KRAS WT and KRAS G13D were constructed and transfected into CLL cell lines MEC1, EHEB and primary CLL cells. The transcriptional levels of CPT1A, CPT2, ACLS1, ACOX1 and HADHA were detected by PCR experiments respectively. As Figure 3 shown, KRAS G13D mutation significantly up-regulated the expression of CPT1A, CPT2 and ACOX1 in CLL cells and down-regulated the expression of ACSL3 in MEC1 cells and EHEB cells (p < 0.05). The expression of HADHA was significantly down-regulated in EHEB cells (p < 0.01), and there was also a downward trend in MEC1 cells.

[0135] As Figure 4 shown, the detection results of primary cells were basically consistent with those of MEC1 cells and EHEB cells. The expressions of CPT1A and ACOX1 were significantly increased in KRAS G13D mutant cells (p < 0.05), and the expressions of ACSL3 and HADHA showed a decreasing trend in KRAS G13D mutant cells. Among them, the expression of ACSL3 was significantly down-regulated in KRAS G13D mutant Pt#1 primary cells (p < 0.05). The above results suggest that compared with KRAS WT CLL cells, fatty acid transport and mitochondrial-dependent fatty acid β-oxidation are enhanced in KRAS G13D mutant CLL cells.

[0136] To clarify the regulatory effect of KRAS G13D mutation on fatty acid oxidation in CLL cells, an extracellular oxygen consumption assay kit was used to detect the oxygen consumption of KRAS WT , KRAS G13D mutant MEC1 and primary CLL cells. It was found that compared with KRAS WT cells, the oxygen consumption of KRAS G13D mutant cells showed an increasing trend. In primary CLL cells, the oxygen consumption of KRAS G13D mutant cells was much higher than that of KRAS WT cells in the later stage ([[]] Figure 5 B-D, p < 0.001), proving that compared with KRAS WT cells, the metabolic energy production of KRAS G13D mutant CLL cells was significantly increased.

[0137] Further, a fatty acid oxidation assay kit was used to detect KRAS WT and KRAS G13D levels of fatty acid oxidation in CLL cells. By comparing the initial slope of fluorescence intensity changes to assess basal fatty acid oxidation activity, it was found that compared with KRAS WT cells, fatty acid oxidation in KRAS G13D cells was significantly enhanced ( Figure 6 , p < 0.01). In summary, KRAS G13D mutation mediates enhanced fatty acid oxidation in CLL cells and promotes the proliferation and survival of CLL cells.

[0138] 2.4 KRAS G13D mutation activates the PI3K pathway in chronic lymphocytic leukemia cells

[0139] To explore the mechanism by which KRAS G13D mutation mediates enhanced fatty acid oxidation levels in CLL cells and thereby promotes the proliferation of CLL cells, MEC1 cells were transfected with KRAS WT and KRAS G13D plasmids respectively. Cells were collected for transcriptome sequencing and differential expression analysis. The results of differential expression analysis are shown in Figure 7 A. Compared with KRAS WT CLL cells, 248 genes were significantly upregulated and 293 genes were significantly downregulated in KRAS G13D -mutated CLL cells, with a total of 541 genes showing significant differences in expression. Among them, genes related to the PI3K / AKT pathway such as PIK3C2A and AKT3, and genes related to tumor lipid metabolism reprogramming such as NGFR, EFNA3, and EGF were significantly upregulated in KRAS G13D -mutated CLL cells. ssGSEA analysis was performed on the gene expression sequences of KRAS WT and KRAS G13D -mutated MEC1 cells respectively. The results showed that compared with KRAS WT cells, there were significant differences in the gene expression of 9 fatty acid metabolism-related pathways such as fatty acid cycle, fatty acid synthesis, long-chain fatty acid transport activity, and unsaturated fatty acid biosynthesis in KRAS G13D -mutated cells ( Figure 7 B). The results of GO enrichment analysis showed that the differentially expressed genes in KRAS G13D -mutated CLL cells were significantly enriched in pathways such as phosphatidylinositol 3-kinase (PI3K) binding, fatty acid derivative binding, and cholesterol storage ( Figure 7 C).

[0140] Result 2.3 has demonstrated through extracellular oxygen consumption experiments and fatty acid oxidation level determination experiments that KRAS G13D mutant CLL cells have significantly enhanced fatty acid oxidation compared to KRAS WT cells. Meanwhile, the results of transcriptome bioinformatics analysis suggest that KRAS G13D mutation upregulates PIK3C2A in CLL cells, potentially activating the PI3K-AKT signaling pathway. Recent studies have found that PI3K-AKT and its downstream effector pathways mediate fatty acid metabolic reprogramming in various tumors, promoting tumorigenesis and progression by regulating fatty acid uptake, synthesis, and oxidation. PIK3C2A encodes the type II PI3K kinase PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2alpha), which is a regulator of membrane trafficking in endocytosis, endosomal recycling, and autophagy, regulating the phosphatidylinositol-3-phosphate (PI3P) lipid pool and lipid membrane morphology. Combining the above research results, it can be speculated that KRAS G13D mutation may promote the proliferation and survival of CLL cells by upregulating the expression of PIK3C2A in CLL cells, mediating the activation of the PI3K-AKT signaling pathway and enhancing fatty acid oxidation. Therefore, to further clarify the regulatory effect of KRAS G13D mutation on the PI3K-AKT signaling pathway in CLL cells, the expression of PIK3C2A in KRAS WT and KRAS G13D mutant CLL cells was detected by PCR, which was consistent with the transcriptome sequencing results. Whether in MEC1 cells ( Figure 8 A), EHEB cells ( Figure 8 B) or primary CLL cells ( Figure 8 C-D), the expression of PIK3C2A in KRAS G13D mutant CLL cells was significantly higher than that in KRAS WT CLL cells. The above results suggest that KRAS G13D mutation upregulates PIK3C2A in CLL cells, activating the PI3K pathway.

[0141] 2.5 KRAS G13D Upregulation of PIK3C2A Activates the PI3K / AKT / mTOR Signaling Pathway in Chronic Lymphocytic Leukemia Cells

[0142] Recent studies have found that the PI3K / Akt / mTOR signaling pathway mediates tumor fatty acid metabolic reprogramming, enhancing metabolic processes such as fatty acid uptake, storage, and cholesterol efflux. Therefore, we regard the PI3K / Akt / mTOR signaling pathway as the main effector pathway by which KRAS G13D mutation upregulates PIK3C2A to promote enhanced fatty acid oxidation in CLL cells. To verify the activation of the PI3K / Akt / mTOR signaling pathway by KRAS G13D mutation in CLL cells, CLL cells with KRAS WT and KRAS G13D mutation were collected, and the protein expressions of PI3K-2α, AKT, phosphorylated AKT, mTOR, and phosphorylated mTOR were detected by Western Blot. As Figure 9 shown, compared with KRAS WT CLL cells, the expressions of PI3K-2α (191 kDa), AKT (60 kDa), mTOR (289 kDa), phosphorylated AKT (56 kDa), and mTOR (289 kDa) were significantly enhanced in KRAS G13D mutant CLL cells. In addition, after treating KRAS G13D CLL cells with the KRAS inhibitor BI-2865 for 24 hours, the protein expressions of the PI3K / AKT / mTOR pathway were detected, and it was found that the expressions of PI3K-2α, phosphorylated AKT, and mTOR were weakened. The above results prove that KRAS G13D mutation activates the PI3K / AKT / mTOR axis in CLL cells, and pharmacological blockade of KRAS G13D activation in CLL cells can inhibit the downstream PI3K / AKT / mTOR signaling pathway.

[0143] To explore the mechanism by which KRAS G13D regulates the activation of PIK3C2A to activate the PI3K / AKT pathway, molecular docking analysis of the proteins encoded by KRAS G13D and PIK3C2A was performed by GRAMM, and it was found that there are multiple KRAS binding sites in the PI3K kinase PI3K-C2α encoded by PIK3C2A, Figure 10 showing the visualization model of the protein complex of the 6 KRAS G13D with the lowest binding energy and PI3K-C2α, suggesting that KRAS G13D can directly bind to the PI3K kinase PI3K-C2α.

[0144] The above studies have clarified the regulatory effect of KRAS G13D mutation on the downstream PI3K / Akt / mTOR signaling pathway, and to further clarify KRAS G13DThe molecular mechanism by which the mutation directly regulates PIK3C2A and then activates the PI3K / Akt / mTOR signaling pathway. The protein interaction between KRAS and PI3K-2α was explored by co-immunoprecipitation (Co-IP) experiment. The results are as Figure 11 shown. In the lysate of KRAS G13D mutant CLL cells, PI3K-C2α (220 kDa) was significantly enriched in the anti-KRAS immunoprecipitation complex, suggesting a protein interaction between KRAS and PI3K-2α. In addition, the KRAS protein enrichment (22 kDa) was also detected in the immunoprecipitation complex captured by the PI3K-2α antibody in the reverse Co-IP, demonstrating the bidirectional specificity of the interaction between KRAS and PI3K-2α. In summary, KRAS G13D mutation activates the PI3K / Akt / mTOR signaling pathway in CLL cells, and there is a structural basis for the direct binding of the KRAS G13D protein to the PI3K-2α encoded by PIK3C2A. The Co-IP experiment confirmed the protein interaction between KRAS and PI3K-2α. KRAS G13D mutation regulates the PI3K / Akt / mTOR signaling pathway in CLL cells by upregulating PIK3C2A, promoting fatty acid oxidation and enhancing the proliferation activity of CLL cells.

[0145] 2.6 Synergistic inhibition of chronic lymphocytic leukemia cell proliferation by KRAS inhibitor and PI3K inhibitor

[0146] PI3K inhibitors have been incorporated into the treatment of CLL with disease progression after receiving Bruton tyrosine kinase (BTK) inhibitor or venetoclax. MIPS-21335 is a PIK3C2A inhibitor (IC50: 0.007 μM), and idelalisib is a PI3Kδ subtype inhibitor (IC50: 0.0025 μM) that has been approved by the US Food and Drug Administration for the treatment of CLL. MEC1, EHEB and primary CLL cells were treated with these two PI3K inhibitors. The drug concentration gradients of MIPS-21335 were 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and the drug concentration gradients of idelalisib were 0 μM, 25 μM, 50 μM, 100 μM, 200 μM. The cell viability was detected by CCK-8 assay after 24 hours of treatment. As Figure 12 shown, the proliferation of CLL cells was inhibited by MIPS-21335 and idelalisib, and the inhibitory effect was concentration-dependent. Under the treatment of the same drug concentration, the viability of KRAS G13D mutant CLL cells was significantly higher than that of KRASWT cells, indicating that compared with KRAS WT cells, the sensitivity of KRAS G13D mutant cells to MIPS-21335 and idelalisib is significantly reduced.

[0147] To investigate the effect of KRAS G13D mutation on the sensitivity of CLL cells to PI3K inhibitor drugs. MEC1, EHEB and primary CLL cells were treated with the PIK3C2A inhibitor MIPS-21335 at a drug concentration of 1 μM, and at the same time, the KRAS inhibitor BI2865 with drug concentration gradients of 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM was added to co-incubate the cells. After 24 hours of treatment, cell viability was detected by CCK-8 assay. As Figure 13 shown, the proliferation of CLL cells was inhibited by the co-treatment of MIPS-21335 and BI2865, and the inhibitory effect was concentration-dependent on BI2865. When MIPS-21335 and BI2865 were used in combination, the viability of KRAS G13D mutant CLL cells was no longer significantly higher than that of KRAS WT cells, indicating that inhibiting KRAS G13D activation promoted the re-enhanced sensitivity of KRAS G13D mutant CLL cells to MIPS-21335, suggesting that CLL patients resistant to PI3K inhibitors may benefit from the combination drug strategy of KRAS inhibitors.

[0148] In KRAS G13D mutant primary Pt#1 cells and MEC1 cells, the drug synergy scores of BI2865 and MIPS-21335 co-administered at concentration gradients of 0, 0.01, 1, and 10 μM were calculated respectively. The drug dose-response was as Figure 14 A and Figure 14 C shown. When MIPS-21335 was 1 μM and BI2865 was 0.01 μM, 0.1 μM, or 1 μM, the drug synergy scores were all greater than 10, indicating that at this combined drug concentration, the two drugs had a synergistic effect on inhibiting the proliferation of CLL cells. Among them, when MIPS-21335 was 1 μM and BI2865 was 0.1 μM, the synergy score was the highest( Figure 14 B, C).

[0149] Matters not covered by this invention are well-known technologies.

[0150] The above embodiments are only for illustrating the technical concept and features of the present invention, and the purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It is not intended to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting KRAS mutations in any one or more of the following: (a1) Preparation of a product for prognostic assessment of chronic lymphocytic leukemia; (a2) Preparation of a product for detecting drug sensitivity of chronic lymphocytic leukemia.

2. The application according to claim 1, characterized in that, The KRAS mutation is KRAS G13D .

3. The application according to claim 1, characterized in that, In (a2), the drug for chronic lymphocytic leukemia is a PI3K inhibitor; further, the PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335.

4. Use of a KRAS mutation or a reagent promoting KRAS mutation in at least one of the following (b1)-(b5): (b1) Upregulating the regulation of the PI3K / AKT / mTOR signaling pathway by PIK3C2A or preparing a product for upregulating the regulation of the PI3K / AKT / mTOR signaling pathway by PIK3C2A; (b2) Enhancing fatty acid oxidation in CLL cells or preparing a product for enhancing fatty acid oxidation in CLL cells; (b3) Promoting the proliferation of CLL cells or preparing a product for promoting the proliferation of CLL cells; (b4) Enhancing the resistance of CLL cells to PI3K inhibitors or preparing a product for enhancing the resistance of CLL cells to PI3K inhibitors; (b5) Constructing a CLL-related disease model.

5. The application according to claim 4, characterized in that The product is a drug or an experimental reagent for non-medical use; The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335; The construction of the CLL-related disease model includes constructing a CLL cell disease model or a CLL animal disease model.

6. Use of a reagent for inhibiting KRAS mutations in at least one of the following (c1)-(c5): (c1) Inhibiting the upregulation of the regulation of the PI3K / AKT / mTOR signaling pathway by PIK3C2A or preparing a product for inhibiting the upregulation of the regulation of the PI3K / AKT / mTOR signaling pathway by PIK3C2A; (c2) Inhibiting fatty acid oxidation in CLL cells or preparing a product for inhibiting fatty acid oxidation in CLL cells; (c3) Inhibiting the proliferation of CLL cells or preparing a product for inhibiting the proliferation of CLL cells; (c4) Inhibiting the resistance of CLL cells to PI3K inhibitors or preparing a product for inhibiting the resistance of CLL cells to PI3K inhibitors; (c5) A product for treating CLL.

7. The application according to claim 6, characterized in that The CLL is KRAS mutant CLL, further including KRAS G13D mutant CLL.

8. The application according to claim 6, wherein The product is a drug or an experimental reagent for non-medical use; The reagent for inhibiting KRAS mutations includes a KRAS inhibitor, specifically BI-2865; The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335.

9. Use of a combination of a KRAS inhibitor and a PI3K inhibitor in the preparation of a drug for treating CLL; The reagent for inhibiting KRAS mutations includes a KRAS inhibitor, specifically BI-2865; The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335; The molar ratio of the KRAS inhibitor to the PI3K inhibitor is 1:0.01 - 10. The CLL is KRAS mutant CLL, further including KRAS G13D mutant CLL.

10. A composition, characterized in that, The composition has active ingredients that at least include a KRAS inhibitor and a PI3K inhibitor; The reagent for inhibiting KRAS mutations includes a KRAS inhibitor, specifically BI-2865; The PI3K inhibitor is the PIK3C2A inhibitor MIPS-21335; The molar ratio of the KRAS inhibitor to the PI3K inhibitor is 1:0.01-10; The composition is used for treating CLL, especially KRAS mutant CLL (including KRAS G13D mutant CLL).

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