Acute leukemia cell strain of human complex karyotype with IKZF1 mutation and application of acute leukemia cell strain

Abstract

The invention discloses a human acute myelogenous leukemia cell strain with positive complex karyotype with IKZF1 mutation as well as a construction method and application of the human acute myelogenous leukemia cell strain. The human acute myelogenous leukemia cell strain with positive complex karyotype with IKZF1 mutation is named as the human acute myelogenous leukemia cell strain YYXY-M9, and is preserved in the China Center for Type Culture Collection (Wuhan University, Wuhan, China) on March 20, 2024, and the preservation number is CCTCC NO: C202482. The leukemia cell strain has a complex karyotype accompanied by IKZF1 mutation and good in-vitro proliferation capacity, and can be used as a cell material for researching a pathogenic mechanism of human acute myelogenous leukemia with the complex karyotype accompanied by IKZF1 mutation positive and in-vitro research of acute myelogenous leukemia with the IKZF1 mutation positive. Meanwhile, the method can be used for drug screening and evaluation research for in-vivo and in-vitro research on human acute myelogenous leukemia with positive human complex karyotype with IKZF1 mutation.

Description

Technical Field

[0001] The invention relates to the fields of biology and oncology, and relates to a human acute myeloid leukemia cell line with normal karyotype, and a construction method and application thereof. Background Art

[0002] Leukemia is the second most common malignant tumor in the blood system after lymphoma, and is also one of the top ten malignant tumors with the highest morbidity and mortality in the world. Among them, acute leukemia has an acute onset, rapid progression, strong heterogeneity, and a worse overall prognosis. Acute myeloid leukemia (AML) is caused by the obstruction of the differentiation and maturation of primitive cells, which leads to the accumulation of primitive cells in the bone marrow or peripheral blood, inhibiting the production of normal blood cells. Clinically, it often manifests as anemia, bleeding, infection and other symptoms. AML often occurs in the elderly, with a median age of about 68 years old. Due to the high heterogeneity of AML, its exact pathogenesis is still unclear. Under current treatment conditions, the overall prognosis of AML is poor, with an overall survival of less than 30% in 5 years. The prognosis of elderly patients and patients with relapsed and refractory disease is even worse, with an overall survival of less than 10% in 5 years. Continuously revealing the pathogenic mechanism of AML, discovering effective therapeutic drugs, such as targeted drugs, optimizing and promoting individualized treatment plans, and ultimately improving the survival of AML are the directions of future efforts.

[0003] With the continuous advancement of biomedical technology, the diagnosis and treatment of AML has gradually shifted towards targeting, individualization and precision. The diagnostic and prognostic system of AML has also evolved from the initial morphology-based to the current comprehensive diagnosis and treatment based on morphology, immunology, cytogenetics and genetics. The role of cytogenetics and molecular biology in the diagnosis, prognosis and risk stratification of AML is becoming increasingly important. The National Cancer Center (NCCN) AML 2023 Guidelines and my country's 2023 AML Expert Consensus both divide AML into three levels based on AML's cytogenetics and molecular biology: good prognosis, moderate prognosis and poor prognosis. Among them, AML with complex karyotype (i.e., the presence of ≥3 chromosomal abnormalities) are classified as poor prognosis group. Studies have confirmed that patients with complex karyotype AML have worse prognosis than AML with other karyotypes.

[0004] In addition to chromosomal karyotype, the significance of molecular biological targets in AML is becoming increasingly clear. For example, AML with PML-Rara has a good prognosis, and with targeted drugs all-trans retinoic acid and arsenic trioxide, AML with PML-RARA fusion gene becomes a non-transplantable and curable acute leukemia type, with a 5-year survival rate of more than 90%. At the same time, AML with FLT3-ITD mutation is further classified into three different prognostic groups according to the expression level of FLT3-ITD. Drugs targeting FLT3 mutation (quizartinib, gilteritinib, etc.) improve the treatment effect of AML with FLT3 mutation. The update of AML treatment guidelines is often accompanied by the inclusion of new molecular targets in the guidelines. Currently, RUNX1 mutation, ASXL1 mutation, SF3B1 mutation, etc. are included in the guidelines. Due to the high heterogeneity of AML, different AML patients have different chromosomal karyotypes and molecular characteristics. Most of the AML-specific chromosomal karyotype characteristics and molecular characteristics have not yet been discovered, and further technological innovation is needed to better understand the molecular characteristics of AML.

[0005] IKZF1 mutation or deletion has been identified in acute lymphoblastic leukemia and has been proven to be a factor in poor prognosis in acute lymphoblastic leukemia. Until recently, different research institutions at home and abroad have discovered that IKZF1 mutations also exist in AML, and the proportion of IKZF1 mutations in AML is between 3% and 5%. AML patients with IKZF1 mutations have also been confirmed to be an independent factor for poor prognosis in AML. There are currently few studies on the mechanism. Cell lines are basic cell materials and tools for studying the causes of tumors, discovering molecular markers, exploring tumor therapeutic drugs and preclinical drug validation, and building animal models. They play an irreplaceable role in promoting tumor research. Although there are many AML cell lines used for research internationally, due to the high heterogeneity of AML's cytogenetics and molecular biology, the cell lines currently used for AML research cannot cover all AMLs with different genetic and molecular characteristics. There are even fewer AML cell lines with Chinese genetic backgrounds established in China. Obviously, it is urgent to establish cell lines that are primary to AML patients and have different molecular characteristics, which is of great significance for studying AMLs with different genetic and molecular characteristics. Since IKZF1 was recently found to play an important role in AML, no AML cell lines with IKZF1 mutations have been established so far, let alone AML cell lines with complex karyotypes.

[0006] In summary, the present invention provides a cell line with a complex karyotype and positive IKZF1 mutation, which is used for the study of IKZF1 mutation AML. Summary of the invention

[0007] The purpose of the present invention is to provide a human complex karyotype cell line with IKZF1 mutation and its construction method and application

[0008] The objective of the present invention is achieved through the following technical scheme: A human complex karyotype cell line with IKZF1 mutation, the cell line is named human acute myeloid leukemia cell line YYXY-M9, the Latin name is Homo sapiens, and it was preserved in the China Center for Type Culture Collection on March 20, 2024, with a preservation number of CCTCC NO: C202482.

[0009] The present invention also provides progeny cells of the human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation as described above.

[0010] The present invention also provides complex karyotype human acute myeloid leukemia cells as described above with IKZF1 mutations. According to the NCCN2023 AML guidelines, AML with complex karyotypes has a poor prognosis, and IKZF1 mutations have recently been confirmed to be an independent poor prognosis molecule for AML, but there are currently no AML cell lines with complex karyotypes and IKZF1 mutations.

[0011] Cell lines are in a suspended state under cell culture conditions, growing individually or in clusters.

[0012] The present invention also provides the use of the human complex karyotype acute myeloid leukemia cell line with IKZF1 mutation as described above, selected from any one or more of the following:

[0013] a. To study the pathogenesis of acute myeloid leukemia with complex karyotype and IKZF1 mutation;

[0014] b. Prepare a tumor cell model or prepare a tumor animal model; wherein, the animal tumor model is prepared by the progeny cells established by this cell line, and the AML animal model is established by subcutaneous tumor loading or tail vein injection modeling.

[0015] c. Screening and / or evaluating / preparing tumor therapeutic drugs; wherein, the method for screening and preparing tumor therapeutic drugs can be: adding different tumor therapeutic drugs to the culture medium of the human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation to obtain effective anti-tumor drugs.

[0016] d. Develop tumor-targeted drugs, including drugs targeting IKZF1 mutations;

[0017] e. Screening tumor biotherapeutic drugs; the tumor biotherapeutic drugs / reagents are tumor vaccines.

[0018] f. Develop tumor-related bioengineering products. The tumor-related bioengineering products can be molecular diagnostic PCR kits and fluorescence in situ hybridization kits for IKZF1 mutations.

[0019] The present invention also provides a method for constructing the complex karyotype acute myeloid leukemia cell line with IKZF1 mutation, comprising the following steps: obtaining 5 ml of fresh peripheral blood from a patient with AML who has been clearly diagnosed, taking a clean and sterile 15 ml centrifuge tube and adding 5 ml of mononuclear cell separation solution to a safety cabinet. The peripheral blood obtained from the AML patient is slowly added along the wall of the centrifuge tube. Then centrifuge at 2000 rpm for 30 minutes. After the centrifugation, use a 1 ml pipette in the safety cabinet to draw the white cell precipitate of the mononuclear cell layer into a new sterile 15 ml sterile centrifuge tube, add 5 ml of sterile 1xPBS pre-cooled in a 4°C refrigerator to resuspend the cells, and centrifuge at 2000 rpm for 5 minutes. After discarding the supernatant, add 5 ml of IMDM complete culture medium (IMDM 90% + fetal bovine serum 10%) to resuspend the cells, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, add 5 ml of IMDM complete culture medium, and resuspend the cells. Count the cells with an automatic cell counter, and take 1x10 8 After adding 10 cells to a T25 cell culture flask, add 6 ml of IMDM complete medium to mix the cells and place them in a 37°C constant temperature and humidity incubator to culture the cells. Depending on the cell proliferation status, replace the IMDM complete medium with new one by centrifugation every 5 to 7 days until the cells begin to proliferate.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] The human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation of the present invention can be infinitely passaged, and the cell shape in vitro is stable, which conforms to the biological characteristics of clinical tumors. The human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation originates from the peripheral blood of AML patients. The human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation can be used for the pathogenic mechanism of complex karyotype and IKZF1 mutation, targeted drug discovery and efficacy evaluation of combined schemes, and can be used to guide clinical drug use. It provides good cell materials for the study of AML with IKZF1 mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with examples and drawings;

[0023] Figure 1 The activity of the human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation under culture conditions and the results after Wright-Giemsa staining;

[0024] Figure 2It is a growth curve diagram of the human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation;

[0025] Figure 3 The chromosome karyotype diagram of the human acute myeloid leukemia cell line with the complex karyotype and IKZF1 mutation;

[0026] Figure 4 It is the Circos diagram of genome variation of whole exome sequencing of human acute myeloid leukemia cell line with complex karyotype and IKZF1 mutation, IKZF1 mutation site and first-generation sequencing verification diagram of mutation site. DETAILED DESCRIPTION

[0027] The present invention is further described below with examples, but the present invention is not limited thereto. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions.

[0028] Example 1 Preparation of YYXY-M4 cell line

[0029] Primary cell culture: Leukemia mononuclear cells were immediately isolated from peripheral blood obtained from a 67-year-old male patient with AML who was hospitalized in the People's Hospital Affiliated to Ningbo University. In a biological safety cabinet, 5 ml of separation solution was added dropwise to a 15 ml sterile centrifuge tube that had been pre-added with 5 ml of mononuclear cell separation solution, and centrifuged at 2000 rpm for 30 minutes. After centrifugation, the mononuclear cell layer was transferred to a new 15 ml sterile centrifuge tube, and 5 ml of sterile 4°C pre-cooled 1xPBS was added to resuspend the cells, and centrifuged at 2000 rpm for 5 minutes. After discarding the supernatant, 5 ml of IMDM complete medium (IMDM 90% + fetal bovine serum 10%) was added to resuspend the cells, and centrifuged at 1500 rpm for 5 minutes. Discard the supernatant, add 6 ml of IMDM complete medium, and resuspend the cells. For the fully automated cell counting plate technology, take 1x10 8 After adding cells to T25 cell culture flask, add IMDM complete medium to 6 ml and mix the cells, then culture the cells in a 37°C constant temperature and humidity incubator. Depending on the cell proliferation status, replace the IMDM complete medium every 5 to 7 days to remove cell debris and continue culturing. Replace the medium every 5 to 7 days until the cells begin to proliferate.

[0030] Cell subculture: Cells begin to proliferate at 8 weeks of culture, growing in suspension or in clusters. At this time, the cell culture medium is replaced every 72 hours and subculture begins. So far, the cells have been subcultured for more than 50 generations, meeting the cell line establishment standards and becoming an immortalized cell line.

[0031] In the present invention, the cell line cells grow in suspension individually or in clusters, the cells are round or oval, and the cell growth rate is stable. It is named YYXY-M9 and was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on March 20, 2024, with the collection number CCTCC NO: C202482.

[0032] Example 2 Biological properties and applications of human acute myeloid leukemia cell lines

[0033] The present invention uses IMDM culture medium containing 10% fetal bovine serum to culture cells YYXY-M9, so that it can grow stably in vitro and be stably passaged. Under a microscope, the cells are observed to grow individually or in clusters, and are round or oval. Wright-Giemsa staining shows that the cells are acute leukemia primitive cells with large and darkly stained nuclei. In vitro proliferation experiments have shown that they can stably proliferate in vitro. Chromosome karyotype analysis shows a complex karyotype, specifically: 47,XY,+2 / 47,XY,+2,t(6;9)(q23;p24) / 47,XY,+2,i(14)(q10) / 46,XY, accompanied by an IKZF1 mutation. The cell line can be used to prepare a tumor cell model with an IKZF1 mutation; to screen and / or evaluate / prepare tumor therapeutic drugs targeting IKZF1; to prepare IKZF1 tumor diagnostic products; and to develop tumor-related bioengineering products. Specifically as follows:

[0034] Morphological observation

[0035] The cultured YYXY-M9 cell line was placed under an inverted microscope to observe whether the cells grew in suspension individually or in clusters. The cells were round or oval in shape. Figure 1 As shown in A. Take 1x10 6 Place the mixture in a 1.5 ml EP tube, centrifuge at 1500 rpm for 5 minutes, discard the supernatant and add 10 μ l medium to resuspend the cells, take 2.510 μ l Cell suspension onto a glass slide, and use another glass slide to slide at a 45-degree angle at a constant speed to make a cell smear. After the cell smear is dry, add Wright-Giemsa stain to stain the cell smear for 5 minutes, rinse, and dry. Observe the cell morphology under an inverted microscope oil lens and take pictures. Figure 1 As shown in B, the cells are mainly primitive cells with large cell size, blue cytoplasm, pale areas around the nucleus, fine nuclear chromatin and obvious nucleoli.

[0036] In vitro proliferation ability observation

[0037] The cultured YYXY-M9 cell line was cultured at 1, 2, 4, 8x10 5 Four cell concentrations of 100 cells / ml were plated in 96-well plates. μl cell solution, add 20 μ l of MTS reagent, and 4 hours later, the absorbance was detected by using an enzyme reader, and the detection wavelength of the enzyme reader was set to 492nm. The proliferation curve of cells at different plating concentrations was drawn using graphpad software. Figure 2 As shown, the cell line cells have good in vitro proliferation ability, and the proliferation rates of cells at different densities are different, and all have in vitro proliferation ability.

[0038] Chromosome karyotype examination

[0039] Count the cultured cells and take 1-2x10 6 Add a certain amount of cell solution to a 15ml centrifuge tube, add colchicine to a final concentration of 0.05μg / ml, shake well and incubate in a 37℃ incubator for 2-3 hours, centrifuge at 1500 rpm / 10min, remove the supernatant, add fixative to fix the cells, the fixative is a 3:1 ratio of methanol and glacial acetic acid, the fixation process is pre-fixation-fixation-fixation, each time with fresh fixative, centrifuge-resuspend, and finally resuspend the cells with the fixative. Then use the chromosome R banding method to develop the bands, analyze manually and take pictures, the results are as follows Figure 3 As shown in AD, the cell line cells have complex karyotype chromosomes, and a total of 4 chromosome karyotypes were detected, namely: 47,XY,+2 / ,;; 47,XY,+2,; t(6;9)(q23;p24); 47,XY,+2,i(14)(q10); 46,XY.

[0040] Whole exome sequencing

[0041] After counting the cultured cells, 5x10 6The cell culture medium was centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, the cell pellets were retained, 1xPBS was added to resuspend the cells and centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cell pellets were retained. The genomic DNA in the sample was extracted, and the quality of the extracted DNA sample was detected by electrophoresis. Then the qualified DNA samples were randomly interrupted into 150bp-220bp fragments by Covaris, and the library was constructed and captured using the Agilent SureSelect Human All Exon V6 kit. The DNA fragments were repaired at the end, ployA tails were added, sequencing adapters were added, purified, magnetic beads were captured, PCR amplified, and other steps were performed to complete the library construction. After the library was qualified, double-end sequencing was performed using a sequencer. After the raw sequencing data (Raw data) was obtained from the sequencing machine, the bioinformatics analysis process was entered, which was divided into two stages: 1. Sequencing data quality assessment: mainly through statistics on sequencing error rate, data volume, alignment rate, coverage, etc., to assess whether the library construction sequencing has reached the standard, and if it meets the standard, subsequent analysis will be performed. 2. Variant information analysis: Align high-quality sequencing sequences to the reference genome, detect variant information in the sample, and analyze and interpret the detected variant information. Figure 4 As shown in A. The SNP site analysis of the YYXY-M9 cell genome revealed IKZF1 mutations, such as Figure 4 As shown in B.

[0042] IKZF1 mutation amplification and validation

[0043] After counting the cultured cells, centrifuge the cell culture medium containing 5x106 at 1500 rpm for 5 minutes, aspirate the supernatant, keep the cell clumps, add 1xPBS to resuspend the cells and centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and keep the cell clumps. According to TAKARA's DNA extraction kit (Cat. No.: CAT#9781), the extraction steps are carried out according to the instructions of the kit. The extracted DNA concentration is measured by a spectrophotometer. TAKARA's PCR reaction kit PrimeSTAR GXL DNA Polymerase (Cat. No.: R050A) is further used to amplify IKZF1, and the 3-step method is used for amplification. For specific steps, please refer to the standard steps of the kit. The present invention also provides primers for amplifying the IKZF1 mutation site, specifically: IKZF1 forward primer IKZF1-F 5'-gcatggcagtggagtaacag-3', IKZF1 reverse primer IKZF1-R 5'-atgcaccacagaataacggc-3'. The PCR reaction product was sent to Shanghai Jierui Biological Company for first-generation sequencing, and the first-generation sequencing results were analyzed by Chromas software. The results are as follows Figure 4As shown in C, the cell line has a SNP change of A<G at the IKZF1 C7:50436033 site.

[0044] Cell STR identification

[0045] The cultured cells were sent to Shanghai Yihe Biological for genotyping of cell STR loci and Amelogenin loci. The results showed that the cell line did not match the existing cell lines in the world and was unique. The genotyping degree of the cell STR loci and Amelogenin loci was 100% with the cell STR loci and Amelogenin loci just isolated from the patient, indicating that they were from the same source, that is, there was no cross contamination during the correct culture process of the cell source. The genotyping of the cell STR loci and Amelogenin loci is shown in Table 1.

[0046] Table 1: Genotyping results of STR loci and Amelogenin loci of cells

[0047]

[0048]

[0049] The above embodiments are used to explain the present invention rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A human acute leukemia cell line with complex karyotype and IKZF1 mutation, characterized in that: The cell line was named human acute myeloid leukemia cell YYXY-M9 and was deposited in the China Center for Type Culture Collection on March 20, 2024, with the preservation number CCTCC NO: 202482.

2. The progeny cell of the human complex karyotype acute leukemia cell line with IKZF1 mutation according to claim 1, characterized in that: The human complex karyotype acute leukemia cell line with IKZF1 mutation is human acute myeloid leukemia cell YYXY-M9.

3. The human acute leukemia cell line with complex karyotype and IKZF1 mutation according to claim 1, characterized in that: The cell morphology of the human acute myeloid leukemia cell YYXY-M9 is primitive cell and the cell chromosome karyotype is a complex karyotype.

4. The human complex karyotype acute leukemia cell line with IKZF1 mutation according to claim 3, characterized in that: The complex karyotype of the human acute myeloid leukemia cell YYXY-M9 is the following four combinations: 47,XY,+2 / 47,XY,+2,t(6;9)(q23;p24) / 47,XY,+2,i(14)(q10) / 46,X, and positive for IKZF1 mutation.

5. The human acute leukemia cell line with complex karyotype and IKZF1 mutation according to claim 3, characterized in that: The human acute myeloid leukemia cells YYXY-M9 grow in a suspended single or clumping state.

6. Use of the human acute leukemia cell line with complex karyotype and IKZF1 mutation as claimed in any one of claims 1 to 5 in the study of the therapeutic mechanism of acute myeloid leukemia with complex karyotype and IKZF1 mutation.

7. Use of the human complex karyotype acute leukemia cell line with IKZF1 mutation as claimed in any one of claims 1 to 5 in preparing a tumor cell model or a tumor animal model.

8. Use of the human acute leukemia cell line with complex karyotype and IKZF1 mutation as claimed in any one of claims 1 to 5 in screening and / or evaluating / preparing tumor therapeutic drugs.

9. Use of the human acute leukemia cell line with complex karyotype and IKZF1 mutation as claimed in any one of claims 1 to 5 in the preparation of tumor vaccines.

10. Use of the human complex karyotype acute leukemia cell line with IKZF1 mutation according to any one of claims 1 to 5 in the preparation of a molecular diagnostic PCR kit for IKZF1 mutation or a fluorescence in situ hybridization kit for IKZF1 mutation.

Images