AML vinca drug-resistant strain and construction method thereof

Abstract

The invention discloses an AML (acute myelogenous leukemia) vinca drug-resistant strain, and belongs to the field of vinca drug-resistant strains, the drug-resistant strain is preserved in the China Center for Type Culture Collection, the preservation name is SKM / Ven, and the preservation number is C2024247; the construction method of the AML vinca drug-resistant strain comprises the following steps: S1, carrying out passage on an initial SKM1 cell in a cell culture device; and S2, adding Vickers with a drug concentration of 2 [mu] M into the cell culture device, treating for 24 hours, rapidly removing dead cells by using a dead cell removal kit, treating by using a drug with a concentration of 2 [mu] M after the state of the cells is recovered, recovering by removing the drug if the number of dead cells is observed, and repeating the operation of adding the drug and removing the drug until the cells stably grow under the concentration, entering the next stage of drug concentration treatment; and S3, according to the drug treatment concentration, selecting a specific drug concentration for progressive increase treatment.

Description

Technical Field

[0001] The present invention relates to the field of venetoclax-resistant strains. Background Art

[0002] Myelodysplastic neoplasms (MDS), also known as myelodysplastic syndrome, are a group of malignant clonal diseases originating from hematopoietic stem / progenitor cells. Their main characteristics are cytopenia, dysplastic hematopoiesis and high risk of transformation to acute myeloid leukemia (AML), called secondary AML (sAML). About 30% of MDS patients will develop sAML within a few months to a few years, and the leukemia transformation rate is higher and faster in high-risk MDS patients than in low-risk MDS patients. Once MDS progresses to AML, the patient's survival time is significantly shortened and the prognosis is poor. Compared with patients with newly diagnosed AML, MDS patients who progress to secondary AML usually have lower response to induction therapy, complete remission rate, relapse-free survival rate and overall survival rate.

[0003] Venetoclax (also known as ABT-199) is a novel targeted drug for the treatment of AML that was approved by the U.S. FDA in 2018 and approved for marketing by the Chinese FDA at the end of 2020. In 2021, my country's latest diagnosis and treatment guidelines added venetoclax combined with demethylating drugs or low-dose cytarabine to low-intensity treatment for patients aged 75 years and above with AML who are not suitable for intensive chemotherapy or have comorbidities, as well as relapsed and refractory AML. It has become a breakthrough new therapy that has profoundly changed the treatment landscape of AML and has broad application prospects. However, in clinical practice, 30-40% of patients develop treatment resistance when they first receive venetoclax treatment, and most patients develop secondary resistance and relapse within months to years after treatment remission. Once resistance occurs, patients will lack effective treatment drugs and their survival time will be shortened. At present, studies have reported the potential resistance mechanism of venetoclax, but there is still a lack of comprehensive analysis of the resistance mechanism of venetoclax. Therefore, resistance to venetoclax is an important cause of ultimate treatment failure and patient death. Studying its resistance mechanism and exploring methods to overcome resistance and restore sensitivity are hot issues and important goals in AML targeted drug research.

[0004] The generation of drug resistance in tumor cells is a complex process, and the establishment of drug-resistant cell lines in vitro is an important means to study the drug resistance mechanism of tumor cells. The method of converting sensitive cell lines to drug-resistant cell lines can be roughly divided into drug concentration increment method, high-dose shock method, high-dose shock combined with concentration increment method, transgenic combined with drug screening method, three-dimensional cell culture method and chemical mutagen, irradiation method, etc. The drug concentration increment method is the most effective and most commonly used method for establishing drug-resistant cell lines. The established drug-resistant cell lines have stable and reliable drug resistance. The established drug-resistant strains can still maintain high drug resistance after freezing or withdrawal of drugs, and the drug resistance multiple is generally higher than the drug-resistant cell lines established in other ways. However, blood tumor cells have the characteristics of suspended growth, and it is impossible to remove dead cells by replacing the culture medium with adherent growth cells. The construction method lacks data reference and has great technical difficulty. At present, there is no relevant report on the construction method of secondary AML venetoclax resistant cell line and its application at home and abroad. The present invention intends to use the drug concentration increment method to construct a typical MDS-to-AML venetoclax resistant cell line. Summary of the invention

[0005] The purpose of the present invention is to address the problems in the prior art and disclose a AML venetoclax-resistant strain, which is deposited in the China Center for Type Culture Collection, with the deposited name: Immortalized resistant cell line SKM / VenHomo sapiens; the deposited number is C2024247, the deposited address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposited date is: August 14, 2024.

[0006] The present application also discloses a method for constructing a AML venetoclax-resistant strain, the steps of which are as follows:

[0007] S1: Subculturing the initial SKM1 cells in a cell culture device;

[0008] S2: Add 2 μM venetoclax to the cell culture device and treat for 24 hours. Use the EasySep Dead Cell Removal Kit to quickly remove dead cells. After the cell state recovers, treat with 2 μM concentration of drug again. If more dead cells are observed, withdraw the drug and the cell culture recovers. Repeat the above "addition-withdrawal" operation until the cells grow stably at this concentration and enter the next stage of drug concentration treatment.

[0009] S3: According to the drug treatment concentration, a specific drug concentration is selected for incremental treatment; the details are as follows:

[0010] When the drug treatment concentration is ≤10 μM, the above “drug addition-drug withdrawal” operation is repeated according to the increasing concentration of 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM;

[0011] When the drug treatment concentration is greater than 10 μM, the concentration is increased in increments of 12 μM, 14 μM, 16 μM, and 18 μM without withdrawing the drug;

[0012] The cells of the "drug resistance induced group" were cultured at least in two groups at the same time, one of which was added with drugs and the other was not. The cells were collected and evenly divided into two groups, and the operation was repeated in this way. As the drug concentration increased, the number of dead cells gradually decreased.

[0013] S4: When the proportion of dead cells stained with trypan blue was ≥50%, the dead cells were removed using a dead cell removal kit; when the proportion of dead cells stained with trypan blue was <50%, the cell debris were removed using low-speed centrifugation;

[0014] S5: After 12 months of induction, the "drug-resistant induced group" was able to stably survive in the presence of 18 μM of the drug, thus obtaining the AML venetoclax-resistant strain.

[0015] In a preferred embodiment, the method for removing dead cells in step S2 is as follows:

[0016] Prepare sterile buffer in advance: prepare PBS buffer containing 2% fetal bovine serum and 1 mM calcium chloride;

[0017] If the total number of cells is less than 1×107, resuspend in 0.1 ml of buffer; if the total number of cells is between 1×107 and 1×108, resuspend in 0.5 ml of buffer and transfer to a 5 ml polystyrene round-bottom tube;

[0018] Add 50 μl / ml Dead Cell Removal Cocktail; add 50 μl / ml Biotin Selection Cocktail, be careful not to vortex the cocktail, gently pipette to mix, and incubate at room temperature for 3 minutes;

[0019] Vortex RapidSpheres for 30 seconds to make the particles evenly dispersed, add 100 μl / ml into the tube, and pipette gently to mix;

[0020] Immediately add culture medium to 2.5 ml, gently pipette to mix, and place the tube in a magnet and incubate at room temperature for 3 min;

[0021] Pick up the magnet and pour into a new sterile centrifuge tube to obtain the required living cells.

[0022] In a preferred embodiment, the centrifugation condition of the low-speed centrifugation method is 500 rpm and 4° C. for 5 min.

[0023] The venetoclax-resistant cell line and transcriptome sequencing data constructed in the present invention can be used for in vivo and in vitro experiments on the venetoclax resistance mechanism of AML, evaluation of drug anti-tumor activity, and development of target drugs for reversing drug resistance, providing an important cell model for the study of the mechanism of venetoclax resistance and having high research and development and production application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of SKM1 cell detection;

[0025] Figure 2 It is the inhibition concentration detection graph;

[0026] Figure 3 It is the cytological characteristics of drug-resistant strains;

[0027] Figure 4 It is the result graph under different concentrations;

[0028] Figure 5 It is a picture of cell apoptosis detection;

[0029] Figure 6 This is the transcriptome level detection diagram of drug-resistant strains;

[0030] Figure 7 A microscopic picture of a cell. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0032] 1. Construction of a secondary AML venetoclax-resistant cell line

[0033] 1. Experimental Materials

[0034] The human MDS-AML transformed cell line SKM1 was introduced and preserved by the Department of Hematology, People's Hospital Affiliated to Jiangsu University, and included in the cell bank of the German National Culture Collection (DSMZ), with the code ACC547. It was correctly identified by STR of Wuhan Pronocell Life Science Co., Ltd. (such as Figure 1 shown).

[0035] 2. Conventional culture method of SKM1 cells

[0036] SKM1 cells are suspension cells and are routinely cultured in 25 cm 2In a permeable cell culture bottle, RPMI-1640 medium (serum: Vicente 086-150; double antibody: Bio-Tec C0222; medium: Vicente 350-006-CL; culture bottle: Labselect 13122) containing 10% fetal bovine serum and 1% antibiotics was placed in a 37°C 5% CO2 incubator. When the cells were in good growth condition, the conventional centrifugation conditions for medium replacement and passage were 4°C 1000 rpm centrifugation for 5 min (Eppendorf refrigerated centrifuge, model Centrifuge 5810R).

[0037] 3. Determination of initial IC50 of SKM1 cells to venetoclax

[0038] SKM1 cells that were correctly identified by STR, in the logarithmic growth phase, and in good condition were prepared into a suspension, and 30,000 cells were inoculated in each well of a 96-well plate, with a volume of 100 μl per well. Each group of treatment was added, and a zero well, a blank control group, and different concentrations of venetoclax treatment groups (the concentration gradient of venetoclax was: 0.025, 0.1, 0.5, 1, 5, 10 μM) (Selleck, S8048) were set up, with 3 replicates per group. To reduce errors, sterile PBS was added to the outermost circle of wells on the culture plate. After the treatment, CCK8 detection was performed, and 10 μl CCK8 solution (TargetMol, C0005) was added to each well. After incubation in the incubator for 2 hours, the absorbance A value of each well was detected at a wavelength of 450 nm using an enzyme reader to ensure that the reading value of the blank control group was between 0.8-1.2. The calculation formula is: cell survival rate = (A value of the experimental group - A value of the zero-adjustment group) / (A value of the control group - A value of the zero-adjustment group) × 100%, and then calculate the half inhibitory concentration (IC50). The experimental results are as follows Figure 2 shown.

[0039] 4. Inducing cell resistance by increasing drug concentration:

[0040] (1) The initial SKM1 cells were subcultured into two cell culture flasks, one as the "parental control group" and the other as the "drug resistance induction group". The two groups of cells were cultured and operated synchronously except for the difference of whether or not drug treatment was added.

[0041] (2) The above experiment obtained that the 24h IC50 of SKM1 to venetoclax was 9 μM. A drug concentration of about 1 / 5 of the initial IC50 (i.e., 2 μM) was used as the starting drug concentration. After adding the drug to the "resistance-inducing group" for treatment for 1 day, the cell status was observed. The mechanism of action of the targeted drug venetoclax is to activate cell apoptosis. Therefore, a large number of dead cells appeared in the "resistance-inducing group" and needed to be removed, especially in the early stage of drug induction. SKM1 is a suspended cell, and dead cells cannot be simply removed by changing the medium of adherent cells. The EasySep Dead Cell Removal Kit (Stemcell, 17899) was used to quickly remove dead cells. The operation steps are as follows:

[0042] ① Prepare sterile buffer in advance: prepare PBS buffer containing 2% fetal bovine serum and 1 mM calcium chloride.

[0043] ② If the total number of cells is less than 1×107, resuspend in 0.1 ml buffer; if the total number of cells is between 1×107 and 1×108, resuspend in 0.5 ml buffer and transfer to a 5 ml polystyrene round-bottom tube (Corning, 352058).

[0044] ③ Add 50 μl / ml Dead Cell Removal (Annexin V) Cocktail; add 50 μl / ml Biotin Selection Cocktail. Be careful not to vortex the cocktail. Mix by gently pipetting and incubate at room temperature for 3 min.

[0045] ④ Vortex RapidSpheres for 30 seconds to make the particles evenly dispersed, add 100 μl / ml into the tube, and pipette gently to mix.

[0046] ⑤Immediately add culture medium to 2.5 ml, gently pipette to mix, and place the tube (without cover) in a magnet (Stemcell, 18000) and incubate at room temperature for 3 min.

[0047] ⑥ Pick up the magnet and pour it into a new sterile centrifuge tube to obtain the required living cells.

[0048] (3) After the cells in the "drug resistance induction group" recover, they are treated with the same concentration of drug again. If more dead cells are observed, the drug is withdrawn and the cells recover. Repeat the above "drug addition-drug withdrawal" operation until the cells grow stably at this concentration, indicating that they can tolerate this concentration and then enter the next higher drug concentration shock. The drug increment is as follows:

[0049] ① When the drug treatment concentration is ≤10 μM, increase the concentration by 2, 4, 6, 8, and 10 μM and repeat the above “drug addition-drug withdrawal” operation.

[0050] ② When the drug treatment concentration is greater than 10 μM, the concentration is increased to 12, 14, 16, and 18 μM, and the drug is not withdrawn. The cells of the "drug resistance induction group" are cultured in two bottles at the same time, one of which is treated with drugs and the other is not treated with drugs. On the second day, the cells are collected and evenly divided into two bottles, and the operation is repeated.

[0051] (4) As the drug tolerance of the "drug resistance induction group" increased, the number of dead cells gradually decreased after increasing the drug concentration. When the proportion of dead cells stained with trypan blue was ≥50%, the dead cells were removed using a dead cell removal kit; when the proportion of dead cells stained with trypan blue was <50%, low-speed centrifugation was used to remove cell debris, and the centrifugation conditions were 500 rpm and 4°C for 5 min.

[0052] (5) After 12 months of induction, the “drug resistance induced group” could stably survive at 18 μM of the drug, indicating that the “drug resistance induced group” could resist the proliferation inhibition caused by venetoclax treatment.

[0053] (6) The obtained "parental control group" was named the parental strain "SKM / Par", and the "resistance induced group" was named the resistant strain "SKM / Ven", and they were preserved in the China Center for Type Culture Collection with the preservation name: immortalized drug-resistant cell line SKM / VenHomo sapiens; the preservation number is C2024247, the preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the preservation date is: August 14, 2024.

[0054] 2. Morphological observation of parental strain and drug-resistant strain

[0055] An inverted optical microscope (Olympus, model CKX53) was used to observe the morphological characteristics of the cells, and modified Wright-Giemsa staining was used to observe changes in the nucleocytoplasm. The cells in the logarithmic growth phase were evenly smeared on the histochemical pen circle of the Cedar high-adhesion slide, fixed with 70% ethanol for 10 minutes, and an appropriate amount of the prepared modified Giemsa staining working solution (1×) (Biyuntian, C0131) was added, stained for 20 minutes, and washed thoroughly from one side with a small stream of tap water. After drying, the cells were observed and photographed under a microscope. Flow cytometry was used to detect the size and granularity of the cells. SKM / Par showed typical cytological characteristics of drug-resistant strains, including irregular morphology, increased volume, increased nuclear-cytoplasmic ratio, and increased intracellular particles. The experimental results are as follows Figure 3 shown.

[0056] 3. Detection of proliferation level of drug-resistant strains

[0057] During the process of drug resistance induction, the 48h IC50 of the "parental control group" and the "resistance induced group" were tested multiple times at intervals to monitor the changes in the resistance index (RI = resistant strain IC50 / parent strain IC50) and the degree of resistance. Compared with the above experimental operation of the initial 24h IC50 of SKM1 cells to venetoclax, the experiment for detecting 48h IC50 has two adjustments: first, the number of cells plated is adjusted to 20,000 cells per well of a 96-well plate to obtain the best CCK8 absorbance value; second, as the drug tolerance of the "resistance-induced group" increases, in order to obtain an accurate IC50 value, the concentration gradient of venetoclax for detecting IC50 should be adjusted accordingly, and the concentration gradient is set to: 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8μM, or 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5, 10, 20μM. The experimental results are as follows Figure 4 As shown, during the process of drug resistance induction, the resistance index of the "resistance induced group" to venetoclax gradually increased from 2.2 times to 6.1 times, which is a moderately resistant strain (5<RI<15 is moderately resistant).

[0058] 4. Detection of apoptosis level of drug-resistant strains

[0059] The collected cell suspension was washed once with pre-cooled PBS and resuspended in 100 μl 1× Binding buffer to adjust the concentration to 1×10 6 / ml, add 5μl PEAnnexinV and 5μl 7-AAD (Novozyme, A213), blow gently, incubate at room temperature in the dark for 10 minutes, add 400μl 1×Bindingbuffer to each well, and complete the detection within 1 hour. Set up a blank control group, PEAnnexinV and 7-AAD single staining group and double staining group, and analyze the results using FlowJo software. The data of three independent repetitions were combined and statistically drawn. The calculation formula is: apoptosis rate (%) = early apoptosis rate (AnnexinV + / PE - )+late apoptosis rate (AnnexinV + / PE + ). The experimental results are as follows Figure 5 As shown, treatment with a certain concentration of venetoclax for 48 hours can significantly increase the apoptosis level of the SKM / Par parental strain, but the same concentration of the drug cannot induce apoptosis in the SKM / Ven resistant strain, indicating that the resistant strain can resist the apoptosis induction of venetoclax.

[0060] 5. Transcriptome level detection of drug-resistant strains

[0061] When the cell culture is in good condition, 1×10 6Each sample was biologically replicated twice, and centrifuged at 4℃1000rpm for 5min. The supernatant was discarded, and 1ml of pre-cooled sterile PBS was added for resuspending. Then the supernatant was discarded and transferred to a 1.5ml EP tube. The cell pellet was quickly frozen in liquid nitrogen for 2min and then subjected to eukaryotic mRNA-seq. Beijing Tiangen Biochemical Technology Co., Ltd. provides library preparation, quality inspection, on-machine sequencing and data analysis services. Illumina sequencing platform PE150 mode sequencing was used to obtain 150bp double-end sequencing reads, and the data volume for each sample was 6GRawdata. After the sequencing raw data was quality controlled, it was referenced to the genome and the sequencing reads were located. The readcount of the transcripts was counted according to the alignment structure, and the expression level and differential expression analysis were calculated. DESeq2 was used to perform differential analysis on the readcount data obtained from the gene expression level analysis. The differential gene screening conditions were |log2(FoldChange)|>1&FDR<0.05 (FoldChange, difference fold; FDR, false discovery rate, i.e., corrected P value). A total of 350 significantly differentially expressed genes were identified in the expression profile of SKM / Ven relative to SKM / Par, including 197 up-regulated genes and 153 down-regulated genes. The experimental results are as follows Figure 6 These differentially expressed genes will provide molecular targets for further studying the mechanism of venetoclax resistance and reversal of SKM1 cells.

[0062] The drug mechanism of Venetoclax is to target and activate the apoptosis pathway. Therefore, during the drug induction process (especially in the early stage), more apoptotic cells, dead cells and dead cell fragments will appear in the "drug resistance induction group" and need to be removed. SKM1 is a suspension cell, and dead cells cannot be removed by replacing the fluid with adherent cells. Since apoptotic cells and dead cells still have a certain weight, it is also difficult to quickly remove them by low-speed centrifugation. If the dead cells cannot be removed in time, the state of drug-tolerant surviving cells will be seriously affected, and the induction time will be greatly prolonged. The present invention is the first to apply the EasySep dead cell removal kit method to the construction of suspension cell resistant strains, which can remove dead cells simply, quickly and accurately. During the process of apoptosis, AnnexinV binds to phosphatidylserine on the cell membrane, and AnnexinV+ apoptotic cells are removed by immunomagnetic negative selection. Unwanted cells are labeled with AnnexinV, antibodies and magnetic particles, and then separated without a column using the EasySep magnet. Finally, the remaining cells are poured into a new test tube to harvest the desired living cells.

[0063] When the proportion of dead cells is high, for example, when the proportion of dead cells is 89.6%, after using the dead cell removal kit, the proportion of dead cells will be reduced to 11.7%, while the low-speed centrifugation method is 88.2%, and the dead cell removal effect is not good; when the proportion of dead cells is low, for example, when the proportion of dead cells is 38.9%, after using the dead cell removal kit, the proportion of dead cells will be reduced to 11.7%, while the low-speed centrifugation method is 14.3%, and the dead cell removal effect is significantly improved. Taking into account the effect and cost, the present invention proposes: when the proportion of dead cells stained with trypan blue is ≥50%, a dead cell removal kit is used to remove dead cells; when the proportion of dead cells stained with trypan blue is <50%, a low-speed centrifugation method is used to remove cell debris, and the centrifugation conditions are 500rpm4℃ centrifugation for 5min. Therefore, the dead cell removal kit is very suitable for the construction of suspended cell resistant strains, especially in the early stage with low drug tolerance and many dead cells.

Claims

1. A venetoclax-resistant strain of AML, characterized in that: The drug-resistant strain is deposited in China Center for Type Culture Collection with the deposit name: SKM / Ven and the deposit number: C2024247.

2. A method for constructing an AML venetoclax-resistant strain as claimed in claim 1, characterized in that: Here are the steps: S1: Subculturing the initial SKM1 cells in a cell culture device; S2: Add 2 μM venetoclax to the cell culture device and treat for 24 hours. Use the EasySep Dead Cell Removal Kit to quickly remove dead cells. After the cell state recovers, treat with 2 μM concentration of drug again. If more dead cells are observed, withdraw the drug and the cell culture recovers. Repeat the above "add drug-withdraw drug" operation until the cells grow stably at this concentration and enter the next stage of drug concentration treatment. S3: According to the drug treatment concentration, a specific drug concentration is selected for incremental treatment; the details are as follows: When the drug treatment concentration is ≤10μM, the above "drug addition-drug withdrawal" operation is repeated according to the increasing concentration of 2μM, 4μM, 6μM, 8μM, and 10μM; When the drug treatment concentration is greater than 10 μM, the concentration is increased in increments of 12 μM, 14 μM, 16 μM, and 18 μM without withdrawing the drug; In the "drug resistance induced group", at least two groups of cells were cultured simultaneously, one of which was treated with drugs and the other was not. The cells were collected and evenly divided into two groups, and the operation was repeated in this way. As the drug concentration increased, the number of dead cells gradually decreased. S4: When the proportion of dead cells stained with trypan blue was ≥50%, the dead cells were removed using a dead cell removal kit; When the proportion of dead cells stained with trypan blue was <50%, low-speed centrifugation was used to remove cell debris; S5: After 12 months of induction, the "drug resistance induced group" was able to stably survive in the presence of 18 μM of the drug, thus obtaining the AML venetoclax-resistant strain.

3. The method for constructing the AML venetoclax-resistant strain according to claim 2, characterized in that: Step S2: The method for removing dead cells is as follows: Prepare sterile buffer in advance: prepare PBS buffer containing 2% fetal bovine serum and 1 mM calcium chloride; If the total number of cells is less than 1 × 10 7 , then resuspend in 0.1 ml buffer; If the total number of cells is between 1×10 7 ~1×10 8 , then resuspend in 0.5 ml buffer and transfer to a 5 ml polystyrene round-bottom tube; Add 50 μl / ml Dead Cell Removal Cocktail; add 50 μl / ml Biotin Selection Cocktail, mix by pipetting, and incubate at room temperature for 3 min; Vortex RapidSpheres for 30 seconds to make the particles evenly dispersed, add 100 μl / ml into the tube, and pipette gently to mix; Immediately add culture medium to 2.5 ml, pipette to mix, and incubate the tube in a magnet at room temperature for 3 min; Pick up the magnet and pour into a new sterile centrifuge tube to obtain the required living cells.

4. The method for constructing the AML venetoclax-resistant strain according to claim 3, characterized in that: The centrifugation conditions of the low-speed centrifugation method are 500 rpm and 4°C for 5 min.

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