Research method for drug resistance of acute T lymphocyte strain to golixitinib

Abstract

The invention discloses a method for researching the drug resistance of an acute T lymphocyte strain to golicotinib, and belongs to the technical field of research on the drug resistance of tumor drugs. According to the method, gradient concentration induction is combined with CRISPR-Cas9 gene editing and screening, a stable golixitinib drug-resistant acute T lymphocyte strain is constructed, a mode of combining multi-omics conjoint analysis and functional verification is adopted, a drug-resistant mechanism is systematically analyzed, and meanwhile, a rapid drug-resistant detection model is established. The invention solves the problems of long construction period of drug-resistant strains, incomplete analysis of drug-resistant mechanism and lack of efficient drug-resistant detection means in the existing research, provides key technical support for clinical medication guidance of golixitinib, research and development of drug-resistant reversal agents and design of novel targeted drugs, and has remarkable clinical application value and industrialization prospect.

Description

Technical Field

[0001] This invention relates to the fields of tumor biology and drug development technology, specifically a method for studying the resistance of acute T lymphocyte lines to golixitinib. Background Technology

[0002] Acute T-lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic malignancy, most commonly seen in children and adolescents, and often faces the challenge of relapse and refractory treatment. Golixitinib, a highly selective JAK3 tyrosine kinase inhibitor, blocks the abnormal proliferation and survival of T-ALL cells by inhibiting the activation of the JAK3-STAT signaling pathway, and has shown good efficacy in clinical studies. However, with prolonged treatment, some patients develop golixitinib resistance, leading to treatment failure, which has become a key bottleneck restricting its clinical application. Existing methods often employ long-term induction at a single concentration, resulting in a construction cycle of 6-12 months. Furthermore, resistant strains exhibit poor stability and are prone to phenotype reversal, failing to meet the needs of subsequent mechanism research and drug screening. Current research focuses solely on JAK3 gene mutations (such as M511I and A634D), neglecting non-genetic mutation resistance mechanisms such as compensatory activation of signaling pathways (e.g., PI3K-AKT and MAPK-ERK pathways), high expression of drug efflux pumps (e.g., ABCG2), and regulation by the tumor microenvironment (e.g., cytokines IL-7 and IL-15). This leads to limitations in our understanding of the nature of drug resistance. Clinically used resistance detection methods rely on cell proliferation inhibition assays (e.g., the CCK-8 assay), which have long detection cycles (requiring 3-5 days) and cannot provide early warning of resistance, hindering timely adjustments to clinical treatment plans. Therefore, those skilled in the art have provided a method for studying acute T lymphocyte resistance to golixitinib to address the problems mentioned in the background. Summary of the Invention

[0003] The purpose of this invention is to provide a method for studying the resistance of acute T lymphocyte lines to golixitinib, in order to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for studying the resistance of acute T lymphocyte lines to golixitinib, comprising the following steps: S1: Construction of glolixitinib-resistant acute T lymphocyte lines: Stable drug-resistant cell lines were constructed by stepwise induction with gradient concentrations combined with single-cell clone screening. S2: Multi-omics analysis of drug resistance mechanisms: Screening drug resistance-related targets and pathways through genomics, transcriptomics, proteomics, phosphorylated proteomics and metabolomics analysis; S3: Functional validation of drug resistance mechanisms: CRISPR-Cas9 gene editing, signaling pathway inhibition, and animal models were used to validate and clarify the functional role of drug resistance mechanisms; S4: Establishment of a rapid drug resistance detection model: Fluorescent immunochromatographic test strips were prepared based on drug resistance biomarkers to achieve rapid drug resistance detection.

[0005] As a further aspect of the present invention: the initial concentration for gradient concentration induction in step S1 is gonixtinib IC50 for acute T lymphocytes. 50 Increase the concentration by 1 / 2 each time, increasing it by 20% until it reaches 5 times the IC50. 50 The induction period is 4-6 weeks.

[0006] As a further aspect of the present invention: the multi-omics joint analysis in step S2 includes: whole exon sequencing to detect gene mutations, RNA-seq to detect differentially expressed genes, TMT quantitative proteomics to detect protein expression differences, phosphorylated proteomics to detect signal pathway activation status, and LC-MS to detect metabolite changes.

[0007] As a further aspect of the present invention, the functional verification in step S3 includes: CRISPR-Cas9-mediated gene mutation knock-in / knockout, combined drug experiment with signaling pathway inhibitors, cytokine regulation experiment, and in vivo verification in a mouse xenograft model.

[0008] As a further embodiment of the present invention: the drug resistance markers in step S4 include JAK3p-M511, ABCG2 protein, and IL7R mRNA, and the detection cycle of the fluorescent immunochromatographic test strip is ≤30 minutes, with a sensitivity ≥90% and a specificity ≥90%.

[0009] As a further embodiment of the present invention: the acute T lymphocyte line is Jurkat cells or CCRF-CEM cells.

[0010] As a further embodiment of the present invention: a golixitinib-resistant acute T lymphocyte line constructed by the above method.

[0011] As a further aspect of the present invention: a golixitinib resistance test strip prepared by the above method.

[0012] As a further aspect of the present invention: the application of drug-resistant acute T lymphocyte lines in the screening of golixitinib resistance reversal agents.

[0013] As a further aspect of the present invention: the application of drug resistance test strips in early warning of golixitinib resistance in patients with acute T-lymphoblastic leukemia.

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. By adopting the strategy of "gradient concentration stepwise induction and single-cell clone screening", the construction cycle is shortened to 4-6 weeks, and the stability of drug-resistant strains is ensured through 15 generations of drug-free passage verification, which solves the problems of long cycle and poor stability of traditional methods.

[0015] 2. For the first time, genomics, transcriptomics, proteomics, phosphorylated proteomics and metabolomics are combined, and the tumor microenvironment regulatory mechanism is also included, so as to achieve a multi-dimensional and systematic analysis of drug resistance mechanism, which breaks through the limitation of traditional focus on gene mutation.

[0016] 3. Based on specific biomarkers screened by multiple omics, a fluorescence immunochromatographic test strip detection model was constructed, which shortened the detection cycle from 3-5 days to less than 30 minutes, enabling early warning of drug resistance and meeting the needs of clinical immediate testing. Attached Figure Description

[0017] Figure 1 This is a graph showing the effect of golixitinib on JURKAT cells; Figure 2 This is a graph showing the effect of golixitinib on CEM cells; Figure 3 This is a Western blot analysis of the expression of STAT1, p-STAT1, STAT3, and p-STAT3 proteins in CEM and jurkat cells at different treatment times. Detailed Implementation

[0018] In this embodiment of the invention, the method for studying the resistance of the acute T lymphocyte cell line to golixitinib includes the following steps: Step 1: Construction of a golixitinib-resistant acute T lymphocyte cell line 1.1 Cell Culture and Initial Drug Sensitivity Testing: Acute T lymphocyte lines in logarithmic growth phase (such as Jurkat cells and CCRF-CEM cells) were used, and the half-maximal inhibitory concentration (IC50) of golixitinib in these cell lines was determined using the CCK-8 assay. 50 The initial induction concentration was determined to be 1 / 2 IC50. 50 ; 1.2 Gradient Concentration Induction and Single-Cell Clone Screening: Cells were divided into experimental and control groups. The experimental group was inducing cell growth at 1 / 2 IC50 concentration. 50 As the starting concentration, change the culture medium containing golixitinib every 3 days, increasing the concentration by 20% each time, until the concentration reaches 5 times the IC50. 50 The control group was cultured in a medium without golixitinib; when the experimental group cells were incubated at 5-fold IC50... 50 After stable proliferation at the concentration for 7 days, single-cell clonal culture was performed using the limiting dilution method to screen and obtain 3-5 single-clonal drug-resistant cell lines; 1.3 Stability verification of drug-resistant strains: The selected monoclonal drug-resistant cell lines were passaged 15 times consecutively in a medium without galixitinib, and the IC50 was measured every 5 passages. 50 Select IC 50 Maintain at more than 5 times the initial IC 50 Cell lines with a coefficient of variation of <10% are designated as stable drug-resistant cell lines (named Res-T-ALL / GOL).

[0019] Step 2: Multi-omics joint analysis of drug resistance mechanisms 2.1 Genomics Analysis: Genomic DNA was extracted from Res-T-ALL / GOL cells and parental cells. Whole-exome sequencing (WES) was used to detect gene mutations, with a focus on analyzing the mutation status of the JAK3 gene and related genes in upstream and downstream signaling pathways (such as STAT5B, PIK3CA, and KRAS). Simultaneously, fluorescence in situ hybridization (FISH) was used to detect JAK3 gene amplification. 2.2 Transcriptomics analysis: RNA-seq technology was used to detect differentially expressed genes between Res-T-ALL / GOL cells and parental cells. GO functional annotation and KEGG pathway enrichment analysis were used to screen for significantly enriched drug resistance-related pathways (such as the PI3K-AKT pathway and drug metabolism pathways). Real-time quantitative PCR (qPCR) was used to verify the expression levels of key differentially expressed genes (such as ABCG2, IL7R, and AKT1). 2.3 Proteomics and Phosphorypomemography Analysis: Tandem mass spectrometry tagging (TMT) quantitative proteomics was used to detect the differences in total protein expression between the two groups of cells; at the same time, phosphorypomemography was used to analyze the changes in phosphorylation levels of key proteins in signaling pathways (such as JAK3, STAT5, AKT, and ERK) to clarify the activation status of the pathways. 2.4 Metabolomics analysis: Liquid chromatography-mass spectrometry (LC-MS) was used to detect the differences in metabolites between the two groups of cells, screen for drug resistance-related metabolic markers (such as lactate and glutamine), and analyze the role of metabolic reprogramming in drug resistance.

[0020] 3.1 Gene Editing Validation: For key mutant genes identified through genomic screening (such as JAK3M511I), the mutation was knocked into parental cells using CRISPR-Cas9 technology. The glycoside concentration (CIC) of the mutant cells was then tested. 50 Changes; simultaneously, knocking out the mutated gene or drug resistance-related genes (such as ABCG2, AKT1) in Res-T-ALL / GOL cells, and detecting whether cellular drug resistance is reversed. The core of the CRISPR-Cas9 system is the specific binding of sgRNA (guide RNA) to the target gene. I. CRISPR-Cas9-mediated JAK1 gene mutation knockout related base sequences: sgRNA1 sequence (20nt): 5'-GCTGCTGCTGCTGCTGCTGC-3' sgRNA2 sequence (20nt): 5'-CGACGACGACGACGACGACG-3' PAM sequence (SpCas9): 5'-NGG-3' (N is any nucleotide, example pairing with sgRNA1: 5'-GCTGCTGCTGCTGCTGCTGCGGG-3') II. CRISPR-Cas9-mediated JAK2 gene mutation knockout related base sequences: sgRNA1 sequence (20nt): 5'-AGAGAGAGAGAGAGAGAGAG-3' sgRNA2 sequence (20nt): 5'-CTCTCTCTCTCTCTCTCT-3' PAM sequence (SpCas9): 5'-NGG-3' (Example paired with sgRNA1: 5'-AGAGAGAGAGAGAGAGAGAGTGG-3') II. CRISPR-Cas9-mediated JAK2 gene mutation knockout related base sequences: sgRNA1 sequence (20nt): 5'-AGAGAGAGAGAGAGAGAGAG-3' sgRNA2 sequence (20nt): 5'-CTCTCTCTCTCTCTCTCT-3' PAM sequence (SpCas9): 5'-NGG-3' (Example paired with sgRNA1: 5'-AGAGAGAGAGAGAGAGAGAGTGG-3') III. CRISPR-Cas9-mediated knock-in of donor DNA (dsDNA) core sequence with JAK1 resistance mutation: Left homologous arm (HA-L, 1000bp fragment, example fragment): 5'-[upstream 1000bp genomic homologous sequence]-3' Drug resistance mutation fragment (example point mutation C→T): 5'-GCTGCTGCTTCTGCTGCTGC-3' Right homologous arm (HA-R, 1000bp fragment, example fragment): 5'-[downstream 1000bp genomic homologous sequence]-3' IV. CRISPR-Cas9-mediated knock-in of donor DNA (dsDNA) core sequence with JAK2 resistance mutation: Left homologous arm (HA-L, 1000bp fragment, example fragment): 5'-[upstream 1000bp genomic homologous sequence]-3' Drug resistance mutation fragment (example point mutation A→G): 5'-AGAGAGAGGGAGAGAGAGAG-3' Right homologous arm (HA-R, 1000bp fragment, example fragment): 5'-[downstream 1000bp genomic homologous sequence]-3'; 3.2 Validation of signaling pathway inhibition: Different signaling pathway inhibitors (such as PI3K inhibitor LY294002 and MAPK inhibitor U0126) were added to Res-T-ALL / GOL cells and treated with golixitinib. The cell proliferation inhibition rate was detected by CCK-8 assay to verify the role of pathway compensatory activation in drug resistance. 3.3 Validation of Tumor Microenvironment Regulation: Different concentrations of cytokines (IL-7, IL-15) were added to the Res-T-ALL / GOL cell culture system, and the cell IC50 was detected. 50 Changes were observed; simultaneously, a mouse xenograft model was constructed, and Res-T-ALL / GOL cells were inoculated into nude mice. Golixitinib was administered alone or in combination with IL-7 / IL-15 neutralizing antibodies, and tumor volume changes were monitored to verify the in vivo drug resistance effect regulated by the microenvironment.

[0021] Step 4: Establishment of a rapid drug resistance detection model 4.1 Screening of drug resistance biomarkers: Based on the results of the multi-omics analysis in step 2, 3-5 specific drug resistance biomarkers were screened. These biomarkers were JAK3p-M511, ABCG2 protein, and IL7R mRNA. 4.2 Preparation of fluorescent immunochromatographic test strips: fluorescent microspheres labeled with marker-specific antibodies (such as anti-JAK3p-M511 antibody and anti-ABCG2 antibody) were used as the detection line; goat anti-mouse IgG antibody was used as the quality control line and fixed on a nitrocellulose membrane to prepare fluorescent immunochromatographic test strips. 4.3 Establishment and Validation of Detection Method: Peripheral blood or bone marrow samples were collected from patients, mononuclear cells were isolated, lysed, and protein samples were obtained. These samples were then added to the sample wells of the test strip. After 15 minutes, the fluorescence intensity ratio (T / C) of the test line and the control line was detected using a fluorescence reader. Res-T-ALL / GOL cell samples were used as positive controls, and parental cell samples were used as negative controls. A standard curve was plotted to determine the threshold for judging drug resistance (T / C > 2.5). Through clinical sample validation, the sensitivity of this test strip can reach 92%, the specificity can reach 95%, and the detection cycle is shortened to less than 30 minutes.

[0022] Example 1: Construction of a Jurkat cell line resistant to glixititinib Initial drug susceptibility testing: Jurkat cells in logarithmic growth phase were seeded in 96-well plates (1×10⁻⁶ cells / wells). 4 Cells / well) were cultured with 0, 0.01, 0.1, 1, 10, and 100 nM globulinib at concentrations of 0, 0.01, 0.1, 1, 10, and 100 nM. After 48 hours of incubation, CCK-8 reagent was added, and the OD450 value was measured to calculate the IC50. 50 The initial induction concentration was determined to be 1.25 nM, with a concentration of 2.5 nM. Gradient induction: Cells in the experimental group were started with 1.25 nM golixitinib, and the medium was changed and the concentration increased by 20% every 3 days, successively to 1.5 nM, 1.8 nM, 2.2 nM, 2.6 nM, 3.1 nM, 3.7 nM, 4.4 nM, 5.3 nM, 6.4 nM, and 7.7 nM (5-fold IC50). 50 After 28 days of culture, the cells proliferated stably. Single-cell clone screening: Cells were seeded into 96-well plates (0.5 cells / well) using the limiting dilution method. After 14 days of culture, 12 single-cell clones were selected, and IC50 was measured. 50 Among them, 3 cell lines had IC 50 >12.5nM; Stability verification: The three cell lines were passaged for 15 generations without drug, and the IC50 was measured every 5 generations. 50 The results showed that the cell line with IC50 value of Res-Jurkat / GOL-2 was positive for IC50. 50 The mM level remained stable at 13.2-14.5 nM with a coefficient of variation of <8%, thus identifying it as a stable drug-resistant cell line.

[0023] Example 2: Elucidation of the Res-Jurkat / GOL Cell Resistance Mechanism Genomic analysis: WES testing revealed the presence of the JAK3 gene M511I mutation in Res-Jurkat / GOL cells, while FISH testing did not detect JAK3 gene amplification; Transcriptomics analysis: RNA-seq screened 218 differentially expressed genes, KEGG enrichment showed significant activation of the PI3K-AKT pathway, and qPCR validation showed that the expression levels of ABCG2, IL7R, and AKT1 mRNA were 3.2-fold, 2.8-fold, and 2.5-fold higher than those in parental cells, respectively. Phosphorylated proteomics analysis: No significant change was detected in the phosphorylation level of JAK3 protein, but the phosphorylation levels of STAT5, AKT, and ERK were 1.8-fold, 2.3-fold, and 1.9-fold higher than those of parental cells, respectively, suggesting compensatory activation of the PI3K-AKT pathway; Metabolomics analysis: LC-MS analysis revealed that the lactate concentration in Res-Jurkat / GOL cells was 2.1 times that of the parental cells, and the glutamine consumption rate was 1.7 times higher, suggesting enhanced glycolysis and glutamine metabolism.

[0024] Example 3: Validation of rapid drug resistance test strips Test strip preparation: Fluorescent microspheres labeled with anti-JAK3p-M511 antibody and anti-ABCG2 antibody were used as the detection line; goat anti-mouse IgG antibody was used as the quality control line and immobilized on a nitrocellulose membrane. Standard curve plotting: Using Res-Jurkat / GOL cell (positive) and Jurkat cell (negative) protein samples as standards, the T / C value was detected, a standard curve was plotted, and the positive threshold was determined to be T / C > 2.5; Clinical sample validation: Samples from 20 T-ALL patients before golixitinib treatment (negative) and 15 resistant patients after treatment (positive) were collected and tested using test strips. The results showed that the sensitivity was 93.3% (14 / 15), the specificity was 95% (19 / 20), and the testing time was 25 minutes.

[0025] The drug-resistant cell lines constructed in this invention have a short cycle (4-6 weeks) and high stability (drug resistance phenotype is not reversed after 15 drug-free passages), solving the efficiency problem of existing construction methods; multi-omics joint analysis enables a comprehensive understanding of drug resistance mechanisms, covering gene mutation, signaling pathway compensation, drug efflux, metabolic reprogramming, and microenvironment regulation, breaking through the limitations of traditional mechanism research; the rapid detection model shortens the detection cycle to less than 30 minutes, with sensitivity and specificity both >90%, meeting the needs of clinical point-of-care testing; It provides ideal cell models and mechanistic targets (such as the PI3K-AKT pathway, ABCG2, and metabolic enzymes) for the development of golixitinib resistance reversal agents; rapid test strips can enable early clinical drug resistance warnings and guide doctors to adjust treatment plans in a timely manner; at the same time, it provides drug resistance mutation information (such as JAK3M511I) for the design of novel JAK3 inhibitors, promoting drug iteration and upgrading.

[0026] The drug-resistant cell lines of this invention can be supplied as commercial cell models, and the rapid test strips can be developed into in vitro diagnostic reagents, showing significant industrialization prospects and generating good economic and social benefits.

[0027] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for investigating the resistance of an acute T lymphocyte cell line to golitinib, characterized by, Comprising the following steps: S1: Construction of golitinib-resistant acute T lymphocyte cell line: stable drug-resistant cell lines are constructed by using gradient concentration step-by-step induction combined with single cell cloning screening; S2: Multi-omics joint analysis of drug resistance mechanism: by genomics, transcriptomics, proteomics, phosphoproteomics and metabolomics analysis, screening drug resistance related targets and pathways; S3: Functional verification of drug resistance mechanism: using CRISPR-Cas9 gene editing, signal pathway inhibition and animal model verification, to clarify the functional role of drug resistance mechanism; S4: Establishment of rapid drug resistance detection model: based on drug resistance markers, fluorescent immunochromatography test strips are prepared to realize rapid detection of drug resistance.

2. The method for researching the resistance of acute T lymphocyte cell lines to golitinib according to claim 1, characterized in that, The starting concentration of the gradient concentration induction in step S1 is 1 / 2 of the acute T lymphocyte cell line Golitnib IC 50 , and each concentration is increased by 20% until reaching 5 times IC 50 , and the induction period is 4-6 weeks.

3. The method of claim 1, wherein the acute T lymphocyte cell line is resistant to golitinib. The multi-omics joint analysis in step S2 comprises: whole exome sequencing to detect gene mutations, RNA-seq to detect differential expression genes, TMT quantitative proteomics to detect protein expression differences, phosphoproteomics to detect signal pathway activation status, and LC-MS to detect metabolite changes.

4. The method of claim 1, wherein the acute T lymphocyte cell line is resistant to golitinib. The functional verification in step S3 comprises: CRISPR-Cas9 mediated gene mutation knock-in / knockout, signal pathway inhibitor combination drug experiment, cytokine regulation experiment and mouse xenograft model in vivo verification.

5. The method of claim 1, wherein the acute T lymphocyte cell line is resistant to golitinib. The drug resistance markers in step S4 include JAK3p-M511, ABCG2 protein and IL7R mRNA.

6. The method of claim 1, wherein the acute T lymphocyte cell line is resistant to golitinib. The acute T lymphocyte cell line is Jurkat cell or CCRF-CEM cell.

7. A golitinib-resistant acute T lymphocyte cell line constructed according to the method of any one of claims 1-6.

8. A golitinib drug resistance detection test strip prepared according to the method of any one of claims 1-6.

9. Use of the drug resistance detection test strip of claim 8 in early warning of golitinib drug resistance in acute T lymphoblastic leukemia patients.

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