CN105986023B - Acute lymphocytic leukemia drug-resistant relapse related mutant gene and application thereof - Google Patents
Acute lymphocytic leukemia drug-resistant relapse related mutant gene and application thereof Download PDFInfo
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Images
Abstract
The invention discloses an acute lymphoblastic leukemia drug-resistant relapse related mutant gene, a kit for evaluating ALL drug-resistant relapse risk and a method for screening drugs for treating or preventing ALL drug-resistant relapse. The mutant gene is a PRPS1 mutant gene. The invention provides powerful technical means and support for preventing and treating drug-resistant relapse of acute lymphoblastic leukemia of children.
Description
Technical Field
The invention relates to the field of biological medicine, and in particular relates to a drug-resistant relapse-related mutant gene of acute lymphocytic leukemia and application thereof.
Background
Acute Lymphoblastic Leukemia (ALL) of children is a common malignant hematological tumor disease in childhood, and is one of the important causes of death of children due to diseases in China. Although treatment and prognosis of childhood ALL have improved significantly over the past few decades, 15% -20% of children suffer relapse, with a cure rate of less than 40% once relapsed, which has always been the leading cause of childhood ALL failure and death. The problem of relapse increasingly becomes a hot spot and focus of ALL research of children at home and abroad.
Chemotherapy is one of the main means of inducing remission and post-remission therapy in the treatment of childhood ALL. The chemotherapy drugs commonly used in clinic at present usually use nucleotide analogs to destroy the synthesis of DNA and RNA, thereby causing DNA damage to induce tumor cell apoptosis. In the ALL induction remission stage of children, 90 percent of patients are remitted by combined chemotherapy of large-dose medicaments, the patients are maintained to be treated by low-dose chemotherapeutic medicaments after remission, and tumor cells need to bear the effect of 6-MP/6-TG to accumulate relapse during the maintenance treatment period mainly by orally taking 6-mercaptopurine (6-MP) or 6-mercaptoguanine (6-TG). Recently, the research group of Adolfo Ferrando and William L Carroll examined samples of drug-resistant relapsed leukemia patients using second-generation sequencing techniques and found that there was a mutation in the 5' -nucleotidase II (NT5C2) gene in relapsed ALL cells, which is responsible for the metabolic-related enzymes encoding 6-mercaptopurine (6-MP) and 6-mercaptoguanine (6-TG) which are the drugs used for nucleotide and nucleotide analogs, with a mutation rate of approximately 10% in ALL drug-resistant relapses. The drug-resistant relapse of children's ALL is not explained by single base mutation of one gene, and the search for genetic variation related to relapse is still the key point and difficulty of the current research on the drug-resistant relapse of children's ALL, and detection and treatment means corresponding to the genetic variation are also lacking.
The PRPS1 gene encodes phosphoribosyl-pyrophosphate synthetase, the first rate-limiting enzyme of the purine metabolic synthesis pathway in cells. PRPS1 catalyzes the reaction of ribose 5-phosphate with ATP to produce AMP and PRPP (ribose 5-phosphate-1-pyrophosphate). PRPP generates hypoxanthine nucleotide (IMP) after a series of enzyme catalytic reactions, and the IMP is further converted into DNA and RNA synthetic raw materials such as (d) ATP and (d) GTP. Too high or too low of PRPP content in cells may cause abnormal metabolic pathways using PRPP as a substrate or a regulator, resulting in epidemic diseases. It has been found that mutation of PRPS1 is associated with diseases such as non-syndromic sensorineural deafness (DFN 2), progressive neuropathic muscular atrophy syndrome (X-linked Charcot-Marie-Tooth disease-5, CMTX5), and the like. PRPS1 mutations have Gain of function mutations (Gain of function), such as D183H, A190V, D52H, etc., and also have loss of function mutations (Reduced function), such as A87T, M115T, I290T, etc. However, no mutation has been found in PRPS1 in tumors to date.
Disclosure of Invention
The technical problems solved by the invention are as follows: aiming at the problem of lack of drug-resistant relapse detection and treatment means of children ALL at present, the invention provides a group of acute lymphoblastic leukemia drug-resistant relapse related mutant gene groups and application thereof in evaluation of children ALL drug-resistant relapse risks. The invention has important guiding significance for subsequent clinical gene diagnosis and individualized treatment in early relapse stage.
The technical scheme for solving the technical problems is as follows:
the invention provides application of PRPS1 as a gene marker of drug-resistant relapse of acute lymphoblastic leukemia.
The invention also provides a PRPS1 mutant, wherein the amino acid sequence of the PRPS1 mutant is formed by the substitution mutation of the following amino acids at the following sites in the sequence shown by SEQ ID No.2 in the sequence table, and the substitution is as follows: serine at position 103 is replaced by threonine, serine at position 103 is replaced by asparagine, asparagine at position 144 is replaced by serine, lysine at position 176 is replaced by asparagine, aspartic acid at position 183 is replaced by glutamic acid, alanine at position 190 is replaced by threonine, leucine at position 191 is replaced by phenylalanine, threonine at position 303 is replaced by serine, valine at position 53 is replaced by alanine, isoleucine at position 72 is replaced by valine, cysteine at position 77 is replaced by serine, aspartic acid at position 139 is replaced by glycine, tyrosine at position 311 is replaced by cysteine, serine at position 103 is replaced by isoleucine, asparagine at position 114 is replaced by aspartic acid or glycine at position 174 is replaced by glutamic acid. The mutant with the serine being replaced with threonine at position 103 is represented as S103T in the invention, and the other mutants are expressed in the same manner, namely S103N, N144S, K176N, L191F, D183E, A190T, T303S, V53A, I72V, C77S, D139G, Y311C, S103I, N114D or G174E.
The invention provides a PRPS1 mutant gene, which encodes the PRPS1 mutant of the amino acid sequence.
In the present invention, the PRPS1 mutant gene may be a gene encoding the above-mentioned PRPS1 mutant, which is a mutant of PRPS1, as is conventional in the art, due to the degeneracy of the nucleotide sequence. The DNA sequence of the mutant gene is preferably formed by the substitution mutation of nucleotides at the following sites in the sequence shown in the sequence table SEQ ID No.1, wherein the substitution is as follows: the method comprises the following steps of replacing a 308 th bit G with C, replacing a 308 th bit G with A, replacing a431 th bit A with G, replacing a 528 th bit G with C, replacing a 549 th bit C with G, replacing a 568 th bit G with A, replacing a 573 th bit G with C, replacing a 908 th bit C with G, replacing a 158 th bit T with C, replacing a214 th bit A with G, replacing a 230 th bit G with C, replacing a416 th bit A with G, replacing a932 th bit A with G, replacing a 308 th bit G with T, and replacing a340 th bit A with G or a 521 th bit G with A.
The 308-position G is replaced by C which is represented as G308C in the invention, and other replacements are similar to the C and are respectively G308A, A431G, G528C, C549G, G568A, G573C, C908G, T158C, A214G, G230C, A416G, A932G, G308T, A340G or G521A.
The invention provides a recombinant vector containing the PRPS1 mutant gene. The recombinant vector is conventional in the art, preferably is a prokaryotic expression vector or a eukaryotic expression vector capable of expressing the PRPS1 mutant gene, more preferably is a lentiviral expression vector capable of expressing the PRPS1 mutant gene, and most preferably is a GV303 lentiviral expression vector (Kjeldahl gene). The preparation method of the eukaryotic expression vector is conventional in the field, and is preferably prepared by the following method: the lentiviral expression vector was linearized with restriction enzymes and passed through In-Fusion by the company clontechTMThe PCR Cloning Kit exchanges the product obtained by PCR amplification of the PRPS1 mutant gene into the linearized lentiviral vector, and the recombinant is amplified through escherichia coli TOP10 to form a recombinant vector containing the PRPS1 mutant gene.
The present invention provides a transformant comprising the recombinant vector. The transformant is conventional in the art, as long as the recombinant vector can stably replicate by itself and the carried PRPS1 mutant gene of the present invention can be effectively expressed, preferably prokaryotic or eukaryotic cells expressing PRPS1 mutant gene of the present invention, more preferably eukaryotic cells expressing PRPS1 mutant gene of the present invention, and most preferably Reh cell line expressing PRPS1 mutant gene of the present invention; the preparation method of the transformant is conventional in the field, and is preferably prepared by packaging a lentivirus vector expressing the PRPS1 mutant gene of the invention into a lentivirus and then infecting the eukaryotic cell, and is most preferably prepared by packaging the lentivirus vector GV303 (Kjek gene) expressing the PRPS1 mutant gene of the invention and a helper plasmid packaged by the virus into a lentivirus, then infecting the eukaryotic cell Reh, and then sorting green fluorescence positive cells by a flow cytometer.
The invention also provides a group of PRPS1 mutant populations for evaluating the risk of drug-resistant relapse of acute lymphoblastic leukemia, wherein the PRPS1 mutant populations comprise the following PRPS1 mutants: the amino acid sequence of the amino acid sequence is shown as SEQ ID No.2 in the sequence table, wherein serine at 103 is replaced by threonine, serine at 103 is replaced by asparagine, asparagine at 144 is replaced by serine, lysine at 176 is replaced by asparagine, aspartic acid at 183 is replaced by glutamic acid, alanine at 190 is replaced by threonine, leucine at 191 is replaced by phenylalanine or threonine at 303 is replaced by serine, namely S103T, S103N, N144S, K176N, L191F, D183E, A190T or T303S.
The population of mutants preferably may also include one or more of the following PRPS1 mutants: the amino acid sequence of the amino acid sequence is shown in SEQ ID No.2 in the sequence table, wherein the 53 th valine is replaced by alanine, the 72 th isoleucine is replaced by valine, the 77 th cysteine is replaced by serine, the 139 th aspartic acid is replaced by glycine, the 311 th tyrosine is replaced by cysteine, the 103 th serine is replaced by isoleucine, the 114 th asparagine is replaced by aspartic acid, the 174 th glycine is replaced by glutamic acid or the 190 th glycine is replaced by valine. Namely V53A, I72V, C77S, D139G, Y311C, S103I, N114D, G174E or a 190V.
The invention provides a PRPS1 mutant gene group for evaluating the drug-resistant relapse risk of acute lymphocytic leukemia, wherein the PRPS1 mutant gene group comprises the following PRPS1 mutant genes: the nucleotide sequence of the nucleotide sequence is shown as SEQ ID No.1 in a sequence table, wherein the 308 th G is replaced by C, the 308 th G is replaced by A, the 431 th A is replaced by G, the 528 th G is replaced by C, the 549 th C is replaced by G, the 568 th G is replaced by A, the 573 th G is replaced by C or the 908 th C is replaced by G. I.e. G308C, G308A, a431G, G528C, C549G, G568A, G573C or C908G.
The mutant gene population preferably may further include one or more of the following PRPS1 mutant genes: the nucleotide sequence of the nucleotide sequence is shown as SEQ ID No.1 in a sequence table, wherein T at position 158 is replaced by C, A at position 214 is replaced by G, G at position 230 is replaced by C, A at position 416 is replaced by G, A at position 932 is replaced by G, G at position 308 is replaced by T, A at position 340 is replaced by G, G at position 521 is replaced by A or C at position 569 is replaced by T. Namely T158C, a214G, G230C, a416G, a932G, G308T, a340G, G521A or C569T.
The invention provides a kit for evaluating ALL drug-resistant relapse risk, which comprises a reagent for detecting a PRPS1 mutant gene in the mutant gene group and an instruction for using.
The reagent may be any reagent conventional in the art for detecting a PRPS1 mutant gene, and preferably includes one or more of a primer, DNA polymerase, dNTP, or buffer for amplifying each exon of PRPS1 gene.
The primer for amplifying each exon of the PRPS1 gene is a primer which is conventional in the field and can amplify each exon of the PRPS1 gene, and preferably the nucleotide sequence of the primer is shown as any one of SEQ ID NO. 3-SEQ ID NO.16 in a sequence table.
The detection kit preferably further comprises a reagent for extracting DNA of the cell or tissue sample, wherein the reagent for extracting DNA of the cell or tissue sample is conventional in the field, and is preferably protease, saturated phenol and the DNA of the cell or tissue sample in a volume ratio of 24: 1, sodium acetate, absolute ethyl alcohol, 70% ethyl alcohol and TE solution, wherein the percentage is volume percentage, and is more preferably a DNA extraction kit produced by Qiagen company.
The DNA polymerase is conventional in the art, and preferably KOD-Plus DNA polymerase manufactured by TOYOBO.
The dNTPs are conventional in the art, and preferably are a mixture of four kinds of dATP, dGTP, dCTP and dTTP.
The buffer solution is a buffer system matched with the DNA polymerase, and is preferably KOD-Plus DNA polymerase buffer solution produced by TOYOBO company.
The instruction book carries the using steps of the kit, and preferably the content of the instruction book comprises the following steps: :
(1) extracting sample genome DNA;
(2) and (2) detecting the PRPS1 mutant gene in the genomic DNA of the sample obtained in the step (1).
In the step (2), the method for detecting the PRPS1 mutant gene in the sample genomic DNA obtained in the step (1) is conventional in the art, and preferably, each exon of the PRPS1 gene is PCR-amplified using the sample genomic DNA obtained in the step (1) as a template, and the amplified fragments are sequenced.
The instructions preferably also include step (3): and detecting that any one or more PRPS1 mutant genes in the PRPS1 mutant gene group exist in the genomic DNA of the sample, so that the sample is at risk of relapse of drug resistance of the acute lymphoblastic leukemia. If the PRPS1 mutant gene exists, the sample is considered to be at risk of drug resistance relapse.
The invention provides a detection method for evaluating the ALL drug-resistant relapse risk of a patient, which comprises the following steps:
(1) extracting sample genome DNA;
(2) and (2) detecting the PRPS1 mutant gene in the genomic DNA of the sample obtained in the step (1).
In step (1), the sample is tumor cells of bone marrow origin or peripheral blood origin or immortalized cell lines cultured in vitro, which are conventional in the art, preferably of the patient to be examined.
In step (1), the genomic DNA of the sample is extracted by conventional methods in the art, preferably by using a DNA extraction kit manufactured by Qiagen.
In the step (2), the method for detecting the PRPS1 mutant gene in the genomic DNA of the sample obtained in the step (1) is conventional in the art, and preferably comprises: and (2) carrying out PCR amplification on each exon of the PRPS1 gene by taking the sample genome DNA obtained in the step (1) as a template, and sequencing the amplified fragments. The PCR amplification is conventional in the art, and is preferably performed using the following conditions:
the PCR system comprises:
PCR specific reaction procedure:
the sequencing is conventional in the art, preferably first-generation sequencing or second-generation sequencing.
The detection method preferably may further include the step (3): and detecting that any one or more PRPS1 mutant genes in the PRPS1 mutant gene group exist in the genomic DNA of the sample, so that the sample is at risk of relapse of drug resistance of the acute lymphoblastic leukemia.
The invention provides a kit for evaluating the drug-resistant relapse risk of acute lymphoblastic leukemia, which comprises the following components: reagents and instructions for use to detect the PRPS1 mutant in the population of PRPS1 mutants.
The reagents are conventional in the art, and are preferably reagents for detecting the enzymatic activity of the PRPS1 mutant, more preferably comprise glucose labeled with a radioisotope carbon atom and PRPP.
The radioisotope carbon atom-labeled glucose is conventional in the art, and is preferably isotopically labeled13C-labeled glucose, said PRPP being conventional in the art, preferably being a PRPP from Sigma company.
The kit preferably further comprises a cell lysis reagent, which is conventional in the art, preferably 80% methanol, said percentages being by volume.
The kit preferably also includes a glucose-free medium, which is conventional in the art, preferably a glucose-free medium available from Gibco.
The instruction is for use of the kit, and the instruction preferably includes the following steps:
(1) lysing the sample cells;
(2) and (3) detecting the PRPS1 mutant in the cell lysate obtained in the step (1).
Wherein the lysis of the sample cells in step (1) is conventional in the art. The method for detecting the PRPS1 mutant in the cell lysate obtained in step (1) in step (2) is conventional in the art, and preferably detects the enzyme activity of PRPS 1. The content described in the specification preferably further includes step (3): when the PRPS1 mutant with higher enzyme activity than the wild-type PRPS1 exists in the sample, the sample has the risk of relapse of the drug resistance of the acute lymphoblastic leukemia. The PRPS1 mutant is preferably any one or more of the PRPS1 mutant populations in (ii).
The invention provides a method for assessing the risk of relapse of ALL resistance in children by detecting PRPS1 enzyme activity, comprising the steps of:
(1) lysing the sample cells;
(2) and (3) detecting the PRPS1 mutant in the cell lysate obtained in the step (1).
Preferably, the method comprises the steps of:
(a) isolating leukemia cells from ALL patients;
(b) culturing the leukemia cells in the step (1);
(c) treating the leukemia cells of step (2) with a chemotherapeutic drug;
(d) collecting the leukemia cells in the step (3), and cracking;
(e) and detecting the PRPP content by LC-MS.
In step (c), the chemotherapeutic agent is conventional in the art, preferably a purine analog chemotherapeutic agent, more preferably 6-MP or 6-TG.
In step (d), the cleavage is conventional in the art, preferably cleavage using 80% methanol.
In step (e), the assay is conventional in the art, preferably the PRPP content is determined using LC-MS.
The invention provides a method for detecting the activity of a medicament for treating or preventing the relapse of ALL resistance in children, which comprises the following steps:
(1) contacting the compound with leukemia cells containing any one of the PRPS1 mutant genes from the PRPS1 mutant gene group to obtain pretreated leukemia cells;
(2) contacting an anti-leukemia chemotherapeutic agent with the pretreated leukemia cells obtained in step (1).
The method preferably may further comprise step (3), step (3) comprising: detecting the survival rate of the cells obtained in the step (2).
The invention provides a method for screening a medicine for treating or preventing drug-resistant relapse of acute lymphoblastic leukemia of children, which comprises the following steps:
(1) contacting a candidate compound with leukemia cells comprising any of the PRPS1 mutant genes from the PRPS1 mutant gene population to obtain pretreated leukemia cells;
(2) contacting an anti-leukemia chemotherapeutic agent with the pretreated leukemia cells obtained in step (1).
In the present invention, the leukemia cells expressing the PRPS1 mutant gene of the present invention in step (1) are conventional in the art, preferably human cells, more preferably human lymphocytes, and most preferably Reh cells or Jurkat cells. The candidate compound is conventional in the field, preferably an existing compound, natural product or nucleic acid drug, more preferably a compound targeting purine synthesis pathway, natural product or nucleic acid drug; the candidate compound is a drug with potential effects of reversing relapse of ALL resistance.
Such contacting is conventional in the art, preferably such that the candidate compound is capable of directly contacting the leukemia cells, more preferably the candidate compound is added directly to the culture medium of the leukemia cells.
In the present invention, the treatment in step (2) is conventional in the art, and preferably the chemotherapeutic agent is allowed to directly contact the leukemia cells, and more preferably the chemotherapeutic agent is directly added to the culture medium of the leukemia cells. The chemotherapeutic drug is preferably a purine drug, and the purine drug is preferably 6-MP or 6-TG.
Preferably, step (1) is also performed without contacting the candidate compound with leukemia cells containing any of the PRPS1 mutant genes from the PRPS1 mutant gene population of claim 9 or 10 as a control.
In the present invention, it is preferable that the method further comprises the step (3) of detecting the survival rate of the cells obtained in the step (2). The detection method in step (3) is conventional in the art, and preferably is a method using a viable Cell dye-stained count method, an MTT method or a method of measuring the intensity of fluorescence emitted by luciferase bound to ATP in viable cells, and more preferably is a method using a commercially available detection kit, such as Cell Titer-Glo reagent from Promega corporation.
Preferably, step (3) is performed while monitoring the viability of the cells in the control of step (2). Survival rates are compared and if the survival rate of the test cells is significantly lower than that of the control cells, the drug to be tested can be considered to have activity to reverse the relapse of ALL resistance.
In the invention, the term "drug-resistant relapse" refers to the occurrence of drug resistance of acute lymphoblastic leukemia patients to the treatment of chemotherapeutic drugs in the convalescent period, wherein the chemotherapeutic drugs are conventional in the field, preferably purine chemotherapeutic drugs, and more preferably 6-mercaptopurine (6-MP) or 6-mercaptoguanine (6-TG).
By "preventing" is meant preventing or reducing the occurrence of relapse to the drug resistance after use, in cases where such relapse is likely to be present.
By "treating" is meant reducing the extent of the relapse of resistance, or curing the relapse of resistance to restore normal status in an acute lymphocytic leukemia patient, or slowing the progression of the relapse of resistance.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the invention provides a mutant gene related to acute lymphocytic leukemia drug-resistant relapse aiming at the problem that the drug-resistant relapse occurs in the treatment of acute lymphocytic leukemia by taking a nucleoside analogue as a chemotherapeutic drug in the prior art, provides a new target spot for the treatment of ALL drug-resistant relapse, can realize the risk evaluation of the drug-resistant relapse of the acute lymphocytic leukemia of children by using the kit for evaluating the ALL drug-resistant relapse risk obtained according to the mutant gene, and provides a powerful technical means and support for the prevention and treatment of the drug-resistant relapse of the ALL of children.
Drawings
FIG. 1: the hyperdeep sequencing found that PRPS1 was a recurrence-specific mutation and the mutation rate increased rapidly before clinical recurrence.
FIG. 2: schematic structure of PRPS1 mutant protein.
FIG. 3: pET28a vector map.
FIG. 4: the purified PRPS1 wild type and each mutant were subjected to SDS-PAGE.
FIG. 5: PCR amplification resulted in PRPS1 wild-type agarose gel electrophoresis.
FIG. 6: western results for stable Reh cell lines expressing PRPS1 wild type and each mutant.
FIG. 7: results of drug sensitivity to 6-MP and 6-TG in stable Reh cell lines expressing PRPS1 wild type and each mutant.
FIG. 8: apoptosis results of stable Reh cell lines expressing PRPS1 wild type and each mutant on 6-MP and 6-TG.
FIG. 9: the detection result of the metabolite of the intracellular chemotherapy drug 6-MP/6-TG.
FIG. 10: the results of enzyme activity detection of PRPS1 expressing PRPS1 wild type and each mutant.
FIG. 11: ADP and GDP are schematic for the PRPS1 feedback regulation path.
FIG. 12: effect of GDP/ADP on the inhibition of PRPS1 protein activity.
FIG. 13: and (5) measuring the content of purine metabolic pathway products in the cells.
FIG. 14: inhibition of resistance of PRPS1S103T and a190T mutants by nucleic acid drugs targeting the purine synthesis pathway.
FIG. 15: exogenous addition of purine affects drug resistance of Reh cells to the chemotherapeutic drug 6-MP.
FIG. 16: exogenous addition of purine affects the metabolism of the chemotherapeutic drug 6-MP by Reh cells.
FIG. 17: hypoxanthine competitively inhibits the response of the chemotherapeutic drug 6-MP.
FIG. 18: lometrexol (Lometrexol) reversed 6-MP drug resistance caused by PRPS1 gene mutation.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
According to the invention, through a whole exome sequencing technology, sequencing is carried out on an initial, remission and relapse sample of 16 groups of children ALL, and the discovery shows that the gene of purine synthesis rate-limiting enzyme PRPS1 (phosphosystematic phosphate synthase I) has relapse specific mutation of multiple sites, and the initial and remission samples are wild types. By further sequencing 144 relapse samples with PRPS1, 16 relapse samples were found to have mutations in the PRPS1 gene, with a mutation frequency of 13% in B-ALL (18/138). At the same time, Charite's medical college in summer in Germany
Berlin) study by the doctor Renate Kirschner-Schwarb at the pediatric hematological tumor center also demonstrated the presence of a recurrence-specific mutation in PRPS1 in german pediatric ALL with a mutation rate of 2.7% (6/220). For the four most prominent mutations of all mutations, A190T, T303S, K176N and N144S, they were found by ultra-deep sequencing of a series of bone marrow samples, and none of the patients in all samples had the above mutations in the resolution range of ultra-deep sequencing during the diagnostic period. In addition, these mutations of PRPS1 were found to increase exponentially before clinical relapse, as shown in fig. 1. The PRPS1 mutant is overexpressed in a PRPS1 wild-type ALL cell line Reh, and drug sensitivity experiments show that the recurrent specific PRPS1 gene mutation enables the Reh to generate drug resistance on 6-MP/6-TG, so that the PRPS1 gene mutation has an important role in the drug resistance recurrence process of children ALL.
The invention combines methods of cell biology, molecular biology, metabonomics and the like, and systematically studies the mechanism of PRPS1 gene mutation mediated children ALL (ALL) resistance relapse from the aspects of the influence of PRPS1 gene mutation on the activity of the enzyme, the influence on 6-MP drug metabolism and purine metabolic network and the like. Research reveals a novel drug-resistant relapse mechanism of childhood ALL, and suggests that PRPS1 mutation can drive relapse, is a gene marker of drug-resistant relapse, and has important guiding significance for subsequent clinical gene diagnosis and individualized treatment in early relapse.
The D183H mutant involved in the following examples is a reported gain-of-function mutant of the PRPS1 gene; the A87T and M115T mutants are reported loss-of-function mutants and are used as positive control and negative control for testing whether the system detected by the experiment is normal or not respectively. The involved S103T, S103N, N144S, T303S, K176N, D183E, a190T, a190V and L191F are experimental groups, and these mutants can be classified into two types by comparing the crystal structure of human PRPS1, i.e., a mutation site located at the dimer formation interface of PRPS1 with a mutation site located at the allosteric site of PRPS1, as shown in fig. 2. The mutants in the group were resistant mutants of the PRPS1 gene.
Example 1
Detecting PRPS1 gene mutation in the sample:
primary, remission and relapse samples and 144 relapse samples of ALL in 16 children were sequenced by whole exome sequencing technique with PRPS 1.
Quality control of the sample: the quality of the sample is directly related to the reliability of the sequencing result, and the quality of the sample for deep sequencing and subsequent verification is ensured by the following three methods: firstly, leukemia cells in bone marrow can account for more than 90 percent of all nucleated cells when leukemia is initially developed and recurs, and residual normal granulocytes and nucleated erythrocytes can be removed through Ficoll density gradient centrifugation, so that high-purity leukemia cells can be obtained for subsequent DNA extraction and sequencing; secondly, leukemia cells and normal bone marrow hematopoietic cells can be distinguished through the combination of multicolor fluorescent antibodies, and high-purity (> 99%) samples can be obtained through the sorting of individual samples with lower purity by a flow cytometry technology; and thirdly, selecting a remission stage sample with residual leukemia cells less than 0.01 percent for deep sequencing according to a trace residual disease detection result, thereby ensuring the reliability of initial and recurrent specific mutation.
Preparation of a sample: extracting leukemia cell genome DNA by QIAGEN Blood DNA kit, measuring DNA concentration by Q-bit fluorescent quantitative kit, and using high-purity genome DNA for deep sequencing at later stage.
Amplification of each exon of PRPS 1: the amplification of each exon of PRPS1 was performed by designing appropriate primers for PRPS1 genomic sequence, and the PCR system and procedure were as follows:
the PCR system comprises:
PCR specific reaction procedure:
the primer sequences are shown in Table 1, and the PCR products are conventionally subjected to capillary electrophoresis sequencing and Mutation analysis using a software of Mutation Surveyor.
Sequencing analysis: after the PCR is successful, sequencing a target band, comparing the target sequence with a PRPS1 gene sequence in NCBI, and analyzing the gene mutation condition of PRPS 1. The results are shown in tables 2 and 3.
By sequencing the initial, remitting and recurrent samples of ALL from 16 groups of children, it was found that there were recurrent specific mutations at multiple sites in the purine synthesis rate-limiting enzyme PRPS1 (phosphorylated phosphate synthase I) gene (see table 2), which were wild-type in both the initial and remitting samples. By sequencing 144 relapse samples with PRPS1, 16 relapse samples were found to have mutation of PRPS1 gene, and the mutation frequency was 13% (18/138). At the same time, Charite's medical college in summer in Germany
Berlin) study of the child hematological tumor center, Roche Kirschner-Schwarb, also confirmed the results of the invention, in Germany childrenThere were recurrent specific mutations in PRPS1 in ALL with a mutation rate of 2.7% (6/220) (see table 2). Combined with clinical pathology analysis, the patients with PRPS1 gene mutation all had early relapse (P)<0.005) (see table 3). The hyperdeep sequencing found that PRPS1 was a recurrence-specific mutation and the mutation rate increased rapidly before clinical recurrence. It means that PRPS1 drives drug-resistant relapse of patients, and can be used as a gene marker for clinical relapse diagnosis and an important target point for clinical relapse treatment.
TABLE 1 PCR amplification exon information
TABLE 2 PRPS1 mutations in B-ALL relapse samples of children in China and Germany
Sequencing ALL initial, remission and relapse bone marrow samples of childhood ALL by a whole exome sequencing technology, and finding that PRPS1 gene mutation exists in relapse samples of 18 patients, wherein the mutation frequency is 13% (18/138), as shown in Table 2. In 9 of the relapse samples, the site of A190T was mutated. By the Charite's medical institute in summer in Germany
Berlin) cooperation with the Renate Kirschner-Schwarb of the pediatric hematological tumor center also demonstrated the presence of a recurrence-specific mutation in PRPS1 in german pediatric ALL with a mutation rate of 2.7% (6/220).
TABLE 3 relationship between PRPS1 mutation and clinical characteristics in B-ALL infants in China and Germany
1The P value is calculated by Fisher's exact test.
2The P value is calculated using the chi-squared test.
As shown in Table 3, through clinical pathology data, PRPS1 mutation is found to occur in early relapsing patients (Chinese patients, P <0.005, Germany patients, P <0.001), indicating that the mutation has the significance of relapse diagnosis in remission stage.
Example 2
Detecting mutation of purine metabolic pathway-associated gene in sample
The purine metabolism-associated enzymes HGPRT, IMPDH, NT5C2, PRPS2, ATIC, ADSL, GART, PFAS were sequenced in 160 samples of ALL relapses from children by conventional secondary sequencing techniques.
Quality control, preparation of samples, and amplification of exons of genes in the same manner as in example 1
Sequencing analysis: and sequencing the target band after the PCR is successful, comparing the target sequence with the corresponding gene sequence in NCBI, and analyzing the gene mutation condition. If the gene mutation is found in the relapse sample, the initial sample of the same patient is detected to determine whether the mutation is a relapse-specific mutation. The results are shown in Table 4.
Through sequencing and sequence alignment, the purine metabolism related enzymes PRPS2, ATIC, ADSL, GART and PFAS have recurrence specificity mutation.
TABLE 4 mutations in purine metabolism-related enzymes in pediatric ALL relapse samples
Example 3
Construction of prokaryotic expression vector PRPS1
1. Obtaining a target gene fragment: the PCR primer sequence for wild-type PRPS1 was designed based on the PRPS1(gene ID:5631, NM002764) sequence provided by NCBI, forward primer: 5 'cgcggcagccatATGCCGAATATCAAAATCTTCAG 3', reverse primer: 5 ' gtggtggtgctcgagTTATAAAGGGACATGGCTGAATAGGTA3 ' (primer description: containing exchange pairing base, enzyme cutting site and target gene 5 ' end part sequence for PCR fishing target gene). The extracted cDNA of the Reh cell is taken as a template, PRPS1 is obtained by PCR, the size of a PCR product is 957bp, and the sequence of the product is shown in a sequence table SEQ ID No. 1.
2. Linearization of prokaryotic expression vectors: pET28a vector was treated with restriction enzymes NdeI/XhoI, and the map of pET28a vector is shown in FIG. 3.
3. Construction of recombinant plasmid: In-Fusion by the company clontechTMThe PCR Cloning Kit was used to transform the PCR product of step 1 into linearized viral vectors and to amplify the recombinants by using E.coli TOP 10.
4. Identifying the recombinant plasmid: whether the recombinants were successfully constructed was verified by sequencing.
Construction of PRPS1 mutant prokaryotic vector: 9 mutants of PRPS1 were constructed by circular PCR with KOD-Plus DNA polymerase from TOYOBO using pET28a-PRPS1 as a plasmid template: S103T, S103N, N144S, T303S, K176N, D183E, A190T, A190V and L191F, and PCR primers are shown in Table 5.
The PCR system comprises:
PCR specific reaction procedure:
the PCR product was digested with 1. mu.l of DpnI enzyme in 37 ℃ water bath for 1 hour, and 10. mu.l of the digested product was amplified by adding to E.coli TOP 10. Sequencing the transformant to verify whether the mutant is successfully constructed.
Example 4
Purification of PRPS1 prokaryotic protein
Escherichia coli BL21 transformed with pET28a-PRPS1 series plasmids (DE3)
[ 1 u l pET28a-PRPS1 series plasmid and 100 u l BL21(DE3) Escherichia coli competent cell mixture, ice placed for 30 minutes, water bath 42 degrees C, 90 seconds, do not shake, immediately placed on ice to cool for 2 minutes, added to 900 u l SOC culture medium, 37 degrees C, 170 rpm, oscillation for 1 hours.
② 1ml of transformed competent cells are smeared on a semisolid LB agar culture dish containing 50 mug/ml kanamycin and are inversely plated for 16 hours, colonies appear, and positive clones with good growth vigor are selected for further experiments.
2. Small-scale expression test:
firstly, selecting a single clone from the pET28a-PRPS1 series transformation plates to 3ml LB (containing antibiotics), and carrying out shaking culture at 37 ℃ and 220rpm for 10-12 hours.
② inoculating bacteria at a ratio of 1:100 the next day, namely taking 50 mul of bacteria liquid to 5ml of LB (containing antibiotics), and carrying out shaking culture at 37 ℃ and 220rpm for 2-3 hours until the OD value reaches 0.6.
③ contrast sampling: 2ml of the bacterial solution was centrifuged at 12000rpm for 1 minute, the supernatant was discarded, 1ml of PBS solution was added to the bacterial pellet, ultrasonication (200w, ultrasonication for 3 seconds, 5 seconds of pause, 20 cycles) was performed, SDS-PAGE Loading Buffer (reduction, 4X) was added, 100 ℃ water bath was performed for 10 minutes, centrifugation was performed at 12000rpm for 10 minutes, and the supernatant was taken and stored at-20 ℃ as a control before induction.
Fourthly, adding an inducer IPTG into the residual 3ml of the bacterial liquid until the final concentration is 1mM, carrying out shaking culture at 16 ℃ and 220rpm for 16 hours, then centrifuging at 12000rpm for 1 minute, discarding the supernatant and collecting the bacteria, carrying out ultrasonic crushing treatment and sampling, and storing at-20 ℃.
Fifthly, carrying out SDS-PAGE electrophoresis on the samples to identify the expression result.
PRPS1 high expression:
firstly, selecting a single clone into 100ml LB (containing 50 mug/ml kanamycin antibiotic), and carrying out shaking culture at 37 ℃ and 220rpm for 10-12 hours.
Expansion culture: inoculating with a ratio of 1:100, namely inoculating the above 20ml bacterial liquid into 2L LB medium (containing kanamycin antibiotic with a final concentration of 50 mug/ml), and performing shake culture at 37 ℃ and 220rpm for 4-5 hours until the OD value reaches 0.6-0.8.
③ contrast sampling: 2ml of the bacterial solution was centrifuged at 12000rpm for 1 minute, the supernatant was discarded, 1ml of PBS solution was added to the bacterial pellet, ultrasonication (200w, ultrasonication for 3 seconds, 5 seconds of pause, 20 cycles) was performed, SDS-PAGE Loading Buffer (reduction, 4X) was added, 100 ℃ water bath was performed for 10 minutes, centrifugation was performed at 12000rpm for 10 minutes, and the supernatant was taken and stored at-20 ℃ as a control before induction.
Fourthly, continuously carrying out shaking culture on the other bacterial liquid at 16 ℃ and 220rpm for 1 hour, adding IPTG (isopropyl-beta-thiogalactoside) to the final concentration of 1mM, carrying out shaking culture at 16 ℃ and 220rpm for 12 hours, and then harvesting: centrifuging at 6000rpm and 4 deg.C for 10 min, discarding supernatant, collecting 5ml for expression identification (16 deg.C sample), and storing at-80 deg.C.
And fifthly, carrying out ultrasonication to respectively process the PRPS1 series samples, and carrying out SDS-PAGE electrophoretic identification. (same as 5 in the small amount expression)
PRPS1 protein purification: respectively taking the ultrasonically-crushed bacterial liquid, purifying the PRPS1 series proteins by a nickel column of an AKTA-purifactor system, identifying the expression and the purity of the PRPS1 series proteins by SDS-PAGE electrophoresis, and quantifying the PRPS1 series proteins by a BCA protein quantification kit of Biyuntan company to obtain the wild type PRPS1 protein of which the amino acid sequence is shown as SEQ ID No.2 in the sequence table, as shown in figure 4.
Example 5
Preparation of PRPS1 mutant gene eukaryotic expression vector
1. Obtaining a target gene fragment: the PCR primer sequence for wild-type PRPS1 was designed based on the PRPS1(gene ID:5631, NM002764) sequence provided by NCBI, forward primer: 5 'GAGGATCCCCGGGTACCGGTCGCCACCATGCCGAATATCAAAATC 3', reverse primer: 5 ' TCCTTGTAGTCCATACCGTGGTGGTGGTGGTGGTGCTCGAGTAAAG3 ', the primer contains exchange pairing basic group, enzyme cutting site and target gene 5 ' end part sequence for PCR. Using pET-28a-PRPS1 as plasmid template, PRPS1 was obtained by PCR, and the size of the PCR product was 1022bp, as shown in FIG. 5.
And (3) PCR system:
PCR reaction procedure:
the PCR product was digested with 1. mu.l of DpnI enzyme in 37 ℃ water bath for 1 hour, and 10. mu.l of the digested product was amplified by adding to E.coli TOP 10. Sequencing the transformant to verify whether the mutant is successfully constructed.
2. Linearization of viral expression vectors: the GV303 viral vector (Gicky gene) was treated with the restriction enzyme AgeI.
3. Construction of recombinant plasmid: In-Fusion by the company clontechTMThe PCR Cloning Kit was used to transfer the PCR product from step 1 into linearized viral vectors according to the instructions and to amplify the recombinant GV303-PRPS1 by E.coli TOP 10.
4. Enzyme digestion identification of recombinant plasmid: whether the recombinants are correct is identified by restriction enzyme Hind III, and if the 354bp enzyme digestion fragment is generated, the recombinants are positive recombinants.
Construction of the PRPS1 mutant plasmid: 9 mutant lentiviral expression vectors of PRPS1 were constructed by circular PCR using the GV303-PRPS1 as a plasmid template and KOD-Plus DNA polymerase from TOYOBO: GV303-PRPS1-S103T, GV303-PRPS1-S103N, GV303-PRPS1-N144S, GV303-PRPS1-T303S, GV303-PRPS1-K176N, GV303-PRPS1-D183E, GV303-PRPS1-A190T, GV303-PRPS1-A190V and GV303-PRPS1-L191F, and the PCR primers are shown in Table 5.
TABLE 5 PRPS1 mutant PCR primer sequence Listing
Example 6
Preparation of Reh cells expressing PRPS1 mutant Gene
Virus preparation
1. HEK293T cells were plated 24 hours before transfection into 10cm dishes and the next day transfection was started when the cell density reached 50% -80%.
2. DNA-Opti-MEM and Fugene-6-Opti-MEM mixtures (one dish of cells) were prepared:
TABLE 6
The transfection reagent Fugene-6 from Promega corporation and Opti-MEM were mixed and left to stand for 5 minutes; mixing the DNA-Opti-MEM and Fugene-6-Opti-MEM, and standing for 15 minutes;
3. and in the process of standing the mixed solution, taking out the HEK293T cell culture dish to be transfected from the incubator, and replacing with a fresh antibiotic-free culture medium.
4. After the mixture was allowed to stand for 15min, 233. mu.l/dish of the mixture was added to a culture dish of HEK293T cells, and the culture solution was mixed uniformly in a cross-shape for 5 times.
Culturing at 5.37 deg.C under 5% CO2, changing culture medium after 24 hr, adding fresh culture medium at a volume of 15 ml/dish, culturing for 72 hr, and collecting virus-containing culture solution.
6. And adding the collected virus supernatant into an Amicon Ultra-15100KD ultrafiltration tube, and centrifuging for 30 minutes at 4 ℃ to obtain concentrated virus liquid.
7. The obtained virus solution was subjected to gradient dilution to infect HEK293T cells, and the virus titer was calculated from the number of cells that fluoresce green after infection.
Viral infection
1. Cell inoculation: the Reh cells were counted and plated in 12-well plates, 3X 10 cells per well5And (4) cells.
2. Concentrated viral supernatant (MOI 10) was added to each well along with polybrene (polybrene) at a final concentration of 8 μ g/ml, and the cells were placed in an incubator for further 24 hours and replaced with fresh complete medium. Fluorescence was observed under a microscope after 48 hours.
3. Flow sorting cells: after 72 hours of virus infection, the cells were separated by a flow cytometer Moflo XDP from Beckman to give Reh cells with green fluorescence, which were then cultured in an expanded manner.
4. And (3) collecting the sorted Reh cells, detecting the expression of the wild type and the mutant type of the PRPS1 through Western blot, and verifying whether the stable cell line is successfully constructed. The results of the experiment are shown in fig. 6, and the following nine stable cell lines were obtained: Reh-PRPS1-S103T, Reh-PRPS1-S103N, Reh-PRPS1-N144S, Reh-PRPS1-T303S, Reh-PRPS1-K176N, Reh-PRPS1-D183E, Reh-PRPS1-A190T, Reh-PRPS1-A190V and Reh-PRPS 1-L191F.
Example 7
6-MP drug susceptibility assay
The Reh cell is a human acute B lymphocyte leukemia strain. The sensitivity of the PRPS1 mutant to the chemotherapeutic drug 6-MP is detected by detecting the survival rate of Reh cells expressing different PRPS1 mutant genes after being treated by the chemotherapeutic drug 6-MP, and the steps are as follows:
inoculating cells: counting Reh cells expressing wild type and various mutant PRPS1, inoculating into 96-well plate, inoculating 10 cells per well4Each cell is provided with 5 multiple holes;
processing cells: diluting the 6-MP with gradient at initial concentration of 100 μ g/ml by 3 times, diluting by 10 gradients, adding into 96-well plate with cells, and culturing at 37 deg.C for 72 hr;
③ measuring the activity of the cells: after 72 hours, 50. mu.l of CellTiter-Glo reagent (Promega corporation) was added to each well, mixed and incubated at room temperature for 10 minutes, and then placed in an enzyme-labeling apparatus (Biotek corporation) to read chemiluminescence values;
fourthly, calculating IC50: drug IC was calculated using Graphpad 5.0 software50Values, compare differences between groups.
The results of the experiment are shown in FIG. 7. Cellular IC of each PRPS1 mutant Gene in the Experimental group compared to the control expressing the empty vector50The values are obviously increased, and the survival rate of the cells expressing various PRPS1 mutant genes in an experimental group is obviously improved after 6-MP treatment, namely the drug resistance is obviously increased.
Example 8
6-TG drug sensitivity test
The sensitivity of different PRPS1 mutants to the chemotherapeutic drug 6-TG is detected by detecting the survival rate of Reh cells expressing different PRPS1 mutant genes after being treated by the chemotherapeutic drug 6-TG, and the steps are as follows:
inoculating cells: counting Reh cells expressing wild type and various mutant PRPS1, inoculating into 96-well plate, inoculating 10 cells per well4Each cell is provided with 5 multiple holes;
processing cells: diluting the 6-TG drug in a gradient manner with the initial concentration of 100 mug/ml, sequentially diluting the drug by 3 times and diluting the drug by 10 gradients, adding the diluted drug into a 96-well plate with well-paved cells, and culturing the drug for 72 hours at 37 ℃;
③ measuring the activity of the cells: after 72 hours, 50. mu.l of CellTiter-Glo reagent (Promega corporation) was added to each well, mixed and incubated at room temperature for 10 minutes, and then placed in an enzyme-labeling apparatus (Biotek corporation) to read chemiluminescence values;
fourthly, calculating IC50: drug IC was calculated using Graphpad 5.0 software50Values, compare differences between groups.
The results of the experiment are shown in FIG. 7. Cellular IC of each PRPS1 mutant Gene in the Experimental group compared to the control expressing the empty vector50Significant increase in value, syndromeObviously, the survival rate of the cells expressing each PRPS1 mutant gene in the experimental group is obviously improved after 6-TG treatment, namely the drug resistance is obviously increased.
Example 9
Detection experiment of apoptosis
Inoculating cells: counting Reh cells expressing wild type and various mutant PRPS1, inoculating into 12-well plate, inoculating 3 × 10 cells per well5Each cell is provided with 2 multiple holes;
processing cells: adding 10 μ g/ml of 6-MP or 6-TG into 12-well-plated cell plate, and culturing at 37 deg.C for 72 hr;
③ dyeing the cells: after 72 hours, centrifuging to collect cells, washing the cells by PBS buffer solution, adding PE-labeled Annexin-V and 7-AAD dye of BD company, uniformly mixing and incubating for 15 minutes at room temperature, centrifuging, removing supernatant, and washing for 2 times by PBS;
flow detection: the apoptosis rate was determined on a flow cytometer Canto II from BD.
The results of the experiment are shown in FIG. 8. Compared with a control expressing no load, the apoptosis ratio of each PRPS1 mutant gene in the experimental group is obviously reduced, and the survival rate of the cells expressing each PRPS1 mutant gene in the experimental group is obviously improved after 6-MP or 6-TG treatment, namely the drug resistance is obviously increased.
Example 10
Detecting the metabolite of the intracellular chemotherapy drug 6-MP/6-TG.
6-MP is a prodrug that needs to undergo metabolic reactions in vivo to form TIMP and TGMP before it can act, as shown in FIG. 9C. The detection steps are as follows:
inoculating cells: reh cells expressing PRPS1 wild type and various mutants were counted in 6cm dishes at 3X 10 cells/dish6A cell;
processing cells: 10 mu M of chemotherapeutic drug 6-MP is added into each 6cm culture dish for treatment for 4 hours;
collecting cells: the cells were transferred to a centrifuge tube, centrifuged at 3,000g for 5 minutes, the supernatant discarded, and the cells were centrifuged at 3X 106Cells were lysed in 200. mu.l 80% methanol;
LC-MS (ABI 5500Qtrap coupled with Waters acquisition UPLC) detects the contents of metabolites TIMP, TGMP, r-MP, r-TG and r-MMP of the chemotherapeutic drug 6-MP, and TIMP (Jean Bioscience, cat # NU-1148), TGMP (Jean Bioscience, cat # NU-1121), r-MP (Sigma, cat #852686), r-TG (Sigma, cat #858412) and r-MMP (Sigma, cat # M4002) are respectively used for quantification by a standard curve.
Experimental results As shown in FIG. 9, for each mutant treated by 6-MP in the experimental group, the intracellular contents of the metabolites TIMP and TGMP of 6-MP, r-TG and r-MMP are all significantly reduced compared with the control.
Example 11
Detection of PRPS1 enzymatic Activity
The invention combines the technology of cell biology and metabonomics, and detects the activity of PRPS1 in cells by an isotope labeling method. The specific implementation mode is as follows:
cell culture: reh cells expressing PRPS1 wild type and various mutants were counted in 6cm dishes at 3X 10 cells/dish6A cell;
processing cells: the following day the cells were suspended in glucose-free medium (Gibco, cat #11879-020) and 10mM each was added to a 6cm dish13C6-labeling with glucose (Cambridge isotopopes laboratories, cat # CLM-1396-1) for 5 minutes;
collecting cells: the cells were transferred to a centrifuge tube, centrifuged at 3,000g for 5 minutes, the supernatant discarded, and the cells were centrifuged at 3X 106Cells were lysed in 200. mu.l 80% methanol;
LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) detection13C5The PRPP content, quantified using a standard curve for PRPP (sigma, cat # P8296).
The experimental results are shown in fig. 10, compared with the control expressing no-load, the concentration values of the catalytic reaction product PRPP of each PRPS1 mutant gene in the experimental group are all obviously increased, and the activity of the PRPS1 mutant enzyme in the cells expressing each PRPS1 mutant gene in the experimental group is proved to be obviously increased.
Example 12
Determination of feedback Regulation of PRPS1 Activity by nucleotides ADP, GDP
1. In vitro protein levels
[ MEANS FOR SOLVING PROBLEMS ] As shown in FIG. 11, an enzymatic reaction catalyzed by PRPS1 was established, and ATP (cat # A7699), ADP (cat # A2754), GDP (cat # G7172), and ribose-5-phosphate (cat # R7750) were purchased from sigma. The reaction system is as follows: 50mM Tris pH7.5, 2mM phosphate, 1mM DTT, 10mM MgCl2, 0.5mM ribose-5-phosphate, 0.5mM ATP, PRPS1 protein.
Measuring the inhibition of GDP/ADP on the activity of PRPS1 protein: GDP/ADP initial concentration is 5mM, and 3 times of concentration gradient dilution is carried out in sequence to prepare 15 uL PRPS1 in-vitro enzymatic reaction system, and the system is added into a 384-well plate to react for 30 minutes at 37 ℃. The reaction was terminated by adding 10. mu.l of Kinase-Glo reagent (cat. No. V3722) from Promega, reacted at room temperature for 15 minutes, and the chemiluminescence value was read on a microplate reader. Inhibition of PRPS1 activity by GDP/ADP was calculated using Graphpad 5.0.
The experimental result is shown in fig. 12A, B, and it can be seen that the inhibition of GDP/ADP on PRPS1 mutant S103T, S103N, N144S, T303S, K176N, D183E, a190T, a190V and L191F is obviously lower than that of PRPS1 wild type, while the known loss-of-function mutation a87T and M115T have no obvious difference from wild type, i.e. the recurrence-specific mutation of PRPS1 escapes the negative feedback inhibition of nucleotide GDP/ADP on the activity of PRPS1, and is a gain-of-function mutation.
2. Cellular level
The invention combines the cell biology and metabonomics technology, detects the activity of PRPS1 in cells by an isotope labeling method, and evaluates the influence of nucleotide GDP/ADP on the activity of PRPS1 on the basis. The specific implementation mode is as follows:
cell culture: reh cells expressing PRPS1 wild type and various mutants were counted in 6cm dishes at 3X 10 cells/dish6A cell;
processing cells: after 4 hours of treatment with 2mM ADP or 0.5mM GDP in the culture dishes, the cells were suspended in a glucose-free medium (Gibco, cat #11879-13C6-labeling with glucose (Cambridge isotopopes laboratories, cat # CLM-1396-1) for 5 minutes;
collecting cells: the cells were transferred to a centrifuge tube, centrifuged at 3,000g for 5 minutes, the supernatant was discarded, as follows3×106Cells were lysed in 200. mu.l 80% methanol;
LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) detection13C5The PRPP content, quantified using a standard curve for PRPP (sigma, cat # P8296). The change of PRPS1 enzyme activity was judged based on the PRPP content.
The experimental result is shown in figure 12C, the concentration of PRPP is reduced after the wild type experimental group is added with the nucleotide GDP/ADP, the concentration of the PRPP which is the catalytic reaction product of each PRPS1 mutant gene in the experimental group is not influenced by the nucleotide GDP/ADP, and the result proves that the activity of PRPS1 mutant enzyme in the cells which express each PRPS1 mutant gene in the experimental group is not regulated by the feedback of the nucleotide GDP/ADP.
Example 13
Detection of purine metabolic pathway Activity
The invention combines the cell biology and metabonomics technology, and detects the activity of a purine de novo synthesis pathway and a salvage synthesis pathway in cells by an isotope labeling method. The specific implementation mode is as follows:
cell culture: reh cells expressing PRPS1 wild type and various mutants were counted in 6cm dishes at 3X 10 cells/dish6A cell;
processing cells: the following day the cells were suspended in amino acid free medium (Gibco custom, cat # ME100031L1) at 20. mu.g/ml each13C2，15N-Glycine (sigma, cat #489522) tags the de novo synthetic pathway, 2. mu.M13C5，15N4-hypoxanthine (Cambridge isotopose laboratories, cat #489522) labeling the salvage synthesis pathway for 4 hours and 1 hour, respectively;
collecting cells: the cells were transferred to a centrifuge tube, centrifuged at 3,000g for 5 minutes, the supernatant discarded, and the cells were centrifuged at 3X 106Cells were lysed in 200. mu.l 80% methanol;
LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) detection13C2， 15N-inosinic acid (IMP +3) and13C5，15N4-content of inosinic acid (IMP +9)A standard curve quantification was performed using IMP (sigma, cat # I4625).
The experimental results are shown in fig. 13, 9B and 18. In Reh cells expressing drug-resistant mutants of each PRPS1 gene in the experimental group13C2， 15N-inosinic acid (IMP +3) and13C5，15N4-the concentration of hypoxanthine nucleotide (IMP +9) and the concentration of hypoxanthine nucleotide are obviously increased compared with Reh cells expressing unloaded and wild-type PRPS1 genes, namely, the activities of two pathways of purine de novo synthesis and salvage synthesis are obviously improved.
Example 14
Detecting the content of hypoxanthine, AICAR and Inosine in the sample
The invention combines the technology of cell biology and metabonomics and detects the content of hypoxanthine in cells by LC-MS. The specific implementation mode is as follows:
cell culture: transfecting human Reh cells with wild type PRPS1 and various mutant viruses (see example 4 for reference), and culturing the cells;
② cell counting: when the cells reach 5X 10 per reaction well6Sampling at 5ml according to the cell number required by the experiment;
collecting cells: cells were aspirated from the wells, centrifuged at 1500rpm and the supernatant discarded. PBS wash 1 time, centrifuge again (500g), aspirate supernatant;
cell lysis: according to 3X 106Cells were lysed in 200. mu.l 80% methanol;
centrifuging at 4 deg.C for 10 min at 14,000g, transferring the supernatant to 1.5ml EP tube;
LC-MS (ABI 5500Qtrap coupled with Waters acquisition UPLC) for detecting the content of hypoxanthine, AICAR and Inosine respectively13C5，15N4Absolute quantification of standard curves for hypoxanthine (Cambridge isotoporate laboratories, cat #489522), AICAR (sigma, cat # A9978) and Inosine (sigma, cat # I4125).
The results of the experiment are shown in FIG. 13. The concentrations of hypoxanthine, AICAR and Inosine in Reh cells expressing drug-resistant mutants of PRPS1 genes in the experimental group were all significantly increased compared to control cells expressing empty and wild-type PRPS1 genes.
Example 15
Nucleic acid drugs targeting purine synthesis pathway
Preparation of lentivirus LV-CRISPR-ATIC and LV-CRISPR-GART aiming at purine de novo synthesis pathway and application of lentivirus LV-CRISPR-ATIC and LV-CRISPR-GART in reversing 6-MP drug tolerance caused by PRPS1 gene mutation
Construction of CRISPR lentiviral vector: according to the CRISPR design principle and the GART and ATIC sequences, the corresponding sequences are respectively designed as follows: TGAATCTGGTCGCTTCCGGA (SEQ ID NO:40) and GCAGCCCGAGTACTTATAAT (SEQ ID NO:39) of CRISPR-GART, and is constructed into a CRISPR lentiviral vector (Addge, cat #49535) to obtain lentivirus vectors lentiCRISPR-ATIC and lentiCRISPR-GART;
② packaging according to a reported method (Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F science 2014Jan 3; 343(6166):84-7.doi:10.1126/science 1247005.Epub 2013Dec 12) to obtain lentiviruses LV-CRISPR-ATIC and LV-CRISPR-GART expressing CRISPR RNA aiming at ATIC and GART genes;
thirdly, infecting cell lines Reh-PRPS1-S103T and Reh-PRPS1-A190T with the packed idle slow virus, slow virus LV-CRISPR-ATIC and LV-CRISPR-GART respectively to form a stable cell line, adding 8 mu g/ml polybrene into the virus MOI which infects Reh cells, changing liquid after infecting 24 hours, culturing for 48 hours, adding 0.8 mu g/ml puromycin for resistance screening for one week to form the following stable cell line: the control cell lines Reh-LV-CRISPR, Reh-S103T-CRISPR-ATIC, Reh-S103T-CRISPR-GART, Reh-A190T-CRISPR-ATIC and Reh-A190T-CRISPR-GART.
Fourthly, cell treatment: diluting the 6-MP, adding into 96-well plate paved with several cell lines, each cell line has 5 multiple wells, detecting the sensitivity change of slow virus infected cell to 6-MP. The specific implementation manner is the same as that of example 8;
the results are shown in FIG. 14, comparing Reh-S103T-CRISPR-ATIC and Reh-S1 with those of the control cell line Reh-LV-CRISPR of unloaded virusIC of 03T-CRISPR-GART, Reh-A190T-CRISPR-ATIC and Reh-A190T-CRISPR-GART50The values all decreased significantly, indicating a significant decrease in resistance to the drug 6-MP.
Example 16
Exogenous purine addition affects drug tolerance of Reh cells to chemotherapeutic 6-MP
The drug sensitivity of externally added purine on Reh cells is detected by the following steps:
inoculating cells: the cells were counted and plated in 96-well plates, 10 cells per well4Each group of the cells is provided with 5 multiple holes;
processing cells: adding Hypoxanthine (HX) or hypoxanthine nucleotide (IMP) of 10 μ M, 50 μ M and 100 μ M to each group respectively for pretreatment for 1 hour, then performing gradient dilution on the medicine 6-MP with the initial concentration of 100 μ g/ml, sequentially diluting 3 times and diluting 10 gradients, adding into a 96-well plate paved with cells, and culturing at 37 ℃ for 72 hours;
③ measuring the activity of the cells: after 72 hours, 50. mu.l of CellTiter-Glo reagent (Promega corporation) was added to each well, mixed and incubated at room temperature for 10 minutes, and then placed in an enzyme-labeling apparatus (Biotek corporation) to read chemiluminescence values;
fourthly, calculating IC50: drug IC was calculated using Graphpad 5.0 software50Values, compare differences between groups.
The results of the experiment are shown in FIG. 15. Compared with purine solvent water control, the experimental group is added with cell IC of hypoxanthine and estramustine nucleotide respectively50The values are obviously increased, and the survival rate of the cells added with the exogenously added purine is obviously improved after 6-MP treatment, namely the drug resistance is obviously increased.
Example 17
Exogenous purine addition influences the metabolism of Reh cells on chemotherapeutic drug 6-MP
Inoculating cells: reh cells were counted and plated on 6cm dishes at 3X 10 cells/dish6A cell;
processing cells: adding 50 μ M Hypoxanthine (HX) or hypoxanthine nucleotide (IMP) into each 6cm culture dish, pretreating for 1 hr, adding 10 μ M chemotherapeutic drug 6-MP, and treating for 4 hr;
collecting cells: the cells were transferred to a centrifuge tube, centrifuged at 3,000g for 5 minutes, the supernatant discarded, and the cells were centrifuged at 3X 106Cells were lysed in 200. mu.l 80% methanol;
LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) detects the contents of the metabolites TIMP and TGMP of the chemotherapeutic 6-MP, and the contents of the metabolites TIMP and TGMP are quantified by respectively using TIMP (Jean Bioscience, cat # NU-1148) and TGMP (Jean Bioscience, cat # NU-1121) as standard curves.
As shown in FIG. 16, for Reh cells treated with exogenously added purine, the intracellular contents of TIMP and TGMP, metabolites of 6-MP, were significantly reduced compared to the control.
Example 18
Hypoxanthine competitively inhibits the response of chemotherapeutic 6-MP
The chemotherapeutics 6-MP is an analogue of hypoxanthine, and 6-MP and hypoxanthine are substrates of HGPRT, and the invention establishes a Km value for determining the reaction of hypoxanthine, 6-MP and HGPRT by in vitro enzymatic reaction and reflects the affinity of hypoxanthine, 6-MP and HGPRT. The specific implementation mode is as follows:
an in vitro enzymatic reaction was set up as shown in figure 17A:
the specific reaction system is as follows:
TABLE 7
| 2*Buffer | Final concentration | Volume (μ l) |
| KCl(mM) | 200 | 320 |
| Tris 8.5(mM) | 200 | 800 |
| MgCl2(mM) | 24 | 96 |
| DTT(mM) | 2 | 8 |
| BSA | 0.01 | 8 |
| H2O was added | 2776 | |
| |
4000 |
TABLE 8
TABLE 9
Sequentially carrying out concentration gradient dilution on hypoxanthine and 6-MP, reacting for 1 hour at 37 ℃, adding 80% methanol to terminate the reaction, and respectively detecting the contents of IMP and TIMP by LC-MS. Reaction curves were plotted and Km values for hypoxanthine and 6-MP were calculated by Graphpad 5.0 software, respectively.
The experimental results are shown in FIG. 17B, where the affinity of hypoxanthine to HGPRT is significantly higher than that of 6-MP to HGPRT; and when 100. mu.M of 6-MP was used as a reaction substrate, the formation of TIMP was significantly inhibited as the concentration of hypoxanthine was increased.
Example 19
Reversal of 6-MP drug tolerance caused by PRPS1 gene mutation by Lometrexol
Lometrexate is a small molecule inhibitor of GART. The invention detects that the lometrexate can reverse 6-MP drug tolerance caused by PRPS1 gene mutation, and the specific steps are as follows:
inoculating cells: counting Reh-PRPS1-S103T and Reh-PRPS1-A190T cells, inoculating into 96-well plate, inoculating 10 cells per well4Each cell line is provided with 5 multiple holes;
processing cells: pretreating cells with 5ng/ml Lometrexol or dimethyl sulfoxide (DMSO) as control for 1 hr, diluting 6-MP in gradient, adding into 96-well plate, and culturing at 37 deg.C for 72 hr;
reading value: after 72 hours, 50. mu.l of CellTiter-Glo reagent (Promega corporation) is added into each hole, mixed and incubated for 10 minutes at room temperature, and placed into an enzyme-linked immunosorbent assay (ELISA) instrument to read chemiluminescence values;
fourthly, calculating: IC calculation Using Graphpad 5.0 software50Value, compare difference;
detecting the concentration of 6-MP metabolite: the same as in example 13;
sixthly, detecting the intracellular concentration of the hypoxanthine: the same as in example 14.
The results of the experiment are shown in FIG. 18. IC of drug 6-MP after treatment of Reh-PRPS1-S103T and Reh-PRPS1-A190T cells with Lometrexol (Lometrexol) compared to respective controls50The values are all remarkably reduced, and simultaneously, the metabolites TIMP and TGMP of the 6-MP in the body are both remarkably increased, which shows that the lometrexate causes that the 6-MP resistance of the cell line of Reh-PRPS1-S103T and Reh-PRPS1-A190T is remarkably reduced. Moreover, the intracellular 6-MP metabolites TIMP and TGMP of Reh-PRPS1-S103T and Reh-PRPS1-A190T after treatment are both increased remarkably, and the treated TIMP and TGMP are also increased remarkablyThe hypoxanthine concentration is obviously reduced.
Example 20
Mass spectrometry detection of purine metabolites and purine analogue drug metabolites
Drugs and agents
Methanol, acetonitrile (HPLC grade) was purchased from Sigma-Aldrich (USA), formic acid (HPLC grade) was purchased from Merck (Germany), and experimental water was prepared from Millipore-Q.
Testing instrument
Liquid chromatograph (UPLC-MS/MS):
AB SCIEX
5500(Singapore)
Waters Ultra Performance LC system(Singapore)
1.ACQUITYTMBinary Solvent Manager
2.ACQUITYTMColumn Manager
3.ACQUITYTMSample Organizer
4.ACQUITYTMSample Manager
an Analyst, version 1.5.2 data acquisition and processing system was used.
Other instruments: thermo Fisher-70 ℃ ultra low temperature refrigerator (usa); eppendorf 5810R high speed large capacity cryogenic centrifuge (germany); IKA Vibrax VXR mini shaker (germany); IKA Vortex oscillator (germany); KQ5200DA ultrasonic cleaner (kunshan).
1. Sample analysis method
Sample processing method
The cells were collected in 80% methanol, centrifuged at 12000rpm at 4 ℃ for 5 minutes, the supernatant was transferred to a new EP tube, and 20. mu.l was taken for LC-MS/MS analysis.
1) Labeled PRPP, ADP and GDP:
chromatographic conditions
Mobile phase composition: mobile phase A: 50mM ammonium bicarbonate (pH9.5)
Mobile phase B: acetonitrile water ═ 6:1(v/v)
Gradient elution:
watch 10
A chromatographic column: ApHeraTM NH2Polymer(2×150mm)；
Flow rate: 0.6 ml/min; sample introduction volume: 20 mu l of the mixture;
mass spectrometric detection conditions
And (3) selecting a multi-channel reaction monitoring (MRM) mode to perform secondary mass spectrometry by adopting an electrospray ion source (Turbo spray). The mass spectrum detection working parameters and ion source parameters are as follows:
TABLE 11
2)IMP(labeled IMP+3and labeled IMP+9),HX,TIMP,TGMP,AICAR,Inosine,6-MP,6-TG,MMP,r-MP,r-TG and r-MMP
Chromatographic conditions
Mobile phase composition: mobile phase A: water-0.025% formic acid-1 mM
Mobile phase B: methanol-0.025% formic acid-1 mM ammonium acetate
Gradient elution:
TABLE 12
A chromatographic column: agilent Eclipse XDB-C18 (4.6X 150mm,5 μm);
flow rate: 0.6 ml/min; sample introduction volume: 15 mu l of the solution;
mass spectrometric detection conditions
And (3) selecting a multi-channel reaction monitoring (MRM) mode to perform secondary mass spectrometry by adopting an electrospray ion source (Turbo spray). The mass spectrum detection working parameters and ion source parameters are as follows:
watch 13
It should be understood that after reading the above description of the present invention, various changes or modifications can be made by those skilled in the art to the relevant conditions of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.
Claims (27)
1. The PRPS1 mutant is characterized in that the PRPS1 mutant is formed by carrying out amino acid substitution mutation on any one of the following sites in a sequence shown as SEQ ID No.2 in a sequence table, wherein the substitution mutation is as follows: serine at position 103 is replaced by threonine, serine at position 103 is replaced by asparagine, asparagine at position 144 is replaced by serine, lysine at position 176 is replaced by asparagine, aspartic acid at position 183 is replaced by glutamic acid, alanine at position 190 is replaced by threonine, leucine at position 191 is replaced by phenylalanine, threonine at position 303 is replaced by serine, valine at position 53 is replaced by alanine, isoleucine at position 72 is replaced by valine, cysteine at position 77 is replaced by serine, aspartic acid at position 139 is replaced by glycine, tyrosine at position 311 is replaced by cysteine, serine at position 103 is replaced by isoleucine, asparagine at position 114 is replaced by aspartic acid or glycine at position 174 is replaced by glutamic acid.
2. A PRPS1 mutant gene, wherein the PRPS1 mutant gene encodes the PRPS1 mutant according to claim 1.
3. The PRPS1 mutant gene of claim 2, wherein the nucleotide sequence of the PRPS1 mutant gene is formed by substitution mutation at any one of the following sites in the nucleotide sequence shown as SEQ ID No.1 of the sequence Listing, and the substitution mutation is: the method comprises the following steps of replacing a 308 th bit G with C, replacing a 308 th bit G with A, replacing a431 th bit A with G, replacing a 528 th bit G with C, replacing a 549 th bit C with G, replacing a 568 th bit G with A, replacing a 573 th bit G with C, replacing a 908 th bit C with G, replacing a 158 th bit T with C, replacing a214 th bit A with G, replacing a 230 th bit G with C, replacing a416 th bit A with G, replacing a932 th bit A with G, replacing a 308 th bit G with T, and replacing a340 th bit A with G or a 521 th bit G with A.
4. A recombinant vector comprising the PRPS1 mutant gene of claim 2 or 3.
5. A transformant, characterized in that it comprises the recombinant vector according to claim 4.
6. A population of PRPS1 mutants for assessing the risk of drug-resistant relapse in acute lymphoblastic leukemia, wherein the population of PRPS1 mutants comprises the following PRPS1 mutants: the amino acid sequence of the amino acid sequence is shown as SEQ ID No.2 in the sequence table, wherein serine at 103 is replaced by threonine, serine at 103 is replaced by asparagine, asparagine at 144 is replaced by serine, lysine at 176 is replaced by asparagine, aspartic acid at 183 is replaced by glutamic acid, alanine at 190 is replaced by threonine, leucine at 191 is replaced by phenylalanine or threonine at 303 is replaced by serine.
7. The population of PRPS1 mutants of claim 6, wherein the population of PRPS1 mutants further comprises one or more of the following PRPS1 mutants: the amino acid sequence of the amino acid sequence is shown in SEQ ID No.2 in the sequence table, wherein the 53 th valine is replaced by alanine, the 72 th isoleucine is replaced by valine, the 77 th cysteine is replaced by serine, the 139 th aspartic acid is replaced by glycine, the 311 th tyrosine is replaced by cysteine, the 103 th serine is replaced by isoleucine, the 114 th asparagine is replaced by aspartic acid, the 174 th glycine is replaced by glutamic acid or the 190 th glycine is replaced by valine.
8. A set of PRPS1 mutant genes for assessing risk of drug-resistant relapse of acute lymphocytic leukemia, wherein the PRPS1 mutant gene group comprises the following PRPS1 mutant genes: the nucleotide sequence of the nucleotide sequence is shown as SEQ ID No.1 in a sequence table, wherein the 308 th G is replaced by C, the 308 th G is replaced by A, the 431 th A is replaced by G, the 528 th G is replaced by C, the 549 th C is replaced by G, the 568 th G is replaced by A, the 573 th G is replaced by C or the 908 th C is replaced by G.
9. The PRPS1 mutant gene population of claim 8, wherein the PRPS1 mutant gene population further comprises one or more of the following PRPS1 mutant genes: the nucleotide sequence of the nucleotide sequence is shown as SEQ ID No.1 in a sequence table, wherein T at position 158 is replaced by C, A at position 214 is replaced by G, G at position 230 is replaced by C, A at position 416 is replaced by G, A at position 932 is replaced by G, G at position 308 is replaced by T, A at position 340 is replaced by G, G at position 521 is replaced by A or C at position 569 is replaced by T.
10. A kit for assessing the risk of drug-resistant relapse in acute lymphoblastic leukemia, comprising: reagents and instructions for use to detect a PRPS1 mutant gene from the PRPS1 mutant gene population of claim 8 or 9.
11. The kit of claim 10, wherein the reagents comprise one or more of primers, DNA polymerase, dntps or buffers for amplifying each exon of PRPS1 gene.
12. The kit according to claim 11, wherein the instructions comprise the steps of:
(1) extracting sample genome DNA;
(2) and (2) detecting the PRPS1 mutant gene in the genomic DNA of the sample obtained in the step (1).
13. The kit according to claim 12, wherein in the step (2), the method for detecting the PRPS1 mutant gene in the genomic DNA of the sample obtained in the step (1) comprises: and (2) carrying out PCR amplification on each exon of the PRPS1 gene by taking the sample genome DNA obtained in the step (1) as a template, and sequencing the amplified fragments.
14. The kit according to claim 12, wherein the instructions further comprise step (3): detecting the presence of one or more PRPS1 mutant genes from the PRPS1 mutant gene population of claim 8 or 9 in genomic DNA of the sample, wherein the sample is at risk of relapse of acute lymphoblastic leukemia drug resistance.
15. The kit of claim 11, wherein the primers for amplifying each exon of the PRPS1 gene are one or more of sequences shown as SEQ ID No.3 to SEQ ID No.16 in the sequence listing; and/or, the kit comprises a reagent for extracting DNA of a cell or tissue sample; and/or the reagent for extracting the genomic DNA of the sample is protease, saturated phenol and a mixture of the protease, the saturated phenol and the saturated phenol in a volume ratio of 24: 1, chloroform and isoamyl alcohol mixed solution, sodium acetate, absolute ethyl alcohol, 70 percent ethyl alcohol and TE solution, wherein the percentage is volume percentage; and/or the reagent for extracting the DNA of the cell or tissue sample is a DNA extraction kit produced by Qiagen company; and/or the DNA polymerase is KOD-Plus DNA polymerase produced by TOYOBO company; and/or the buffer solution is KOD-Plus DNA polymerase buffer solution produced by TOYOBO company; and/or the dNTP is a mixture of four kinds of dATP, dGTP, dCTP and dTTP.
16. A kit for assessing the risk of drug-resistant relapse in acute lymphoblastic leukemia, comprising: reagents and instructions for detecting a PRPS1 mutant in a population of PRPS1 mutants according to claim 6 or 7.
17. The kit of claim 16, wherein the reagent is a reagent for detecting the enzymatic activity of PRPS1 mutant.
18. The kit of claim 17, wherein the reagents comprise PRPP and glucose labeled with a radioisotope carbon atom.
19. The kit of claim 18, wherein the glucose labeled with a radioisotope carbon atom is13C-labeled glucose.
20. The kit of claim 16, wherein the instructions comprise the steps of:
(1) lysing the sample cells;
(2) and (3) detecting the PRPS1 mutant in the cell lysate obtained in the step (1).
21. The kit according to claim 20, wherein the instructions further comprise step (3): detecting that PRPS1 mutant with higher enzyme activity than wild-type PRPS1 exists in the sample, and then the sample has the risk of drug resistance relapse of acute lymphoblastic leukemia; and/or the PRPS1 mutant with higher enzymatic activity than wild-type PRPS1 is any one or more of the PRPS1 mutant population as claimed in claim 6 or 7.
22. The kit of claim 20, wherein the step (2) of detecting the PRPS1 mutant in the cell lysate of step (1) is detecting the enzymatic activity of PRPS 1.
23. The kit of claim 16, wherein the kit further comprises a cell lysis reagent; and/or, the cell lysis reagent is 80% methanol, the percentage is volume percentage; and/or, the kit further comprises a glucose-free medium; and/or the glucose-free medium is a glucose-free medium purchased from Gibco, Inc.
24. A method for detecting the activity of a compound for treating or preventing relapse of acute lymphoblastic leukemia resistance in children, comprising the steps of:
(1) contacting a compound with Reh cells or Jurkat cells comprising a PRPS1 mutant gene from any of the PRPS1 mutant gene populations of claim 8 or 9 to obtain preconditioned Reh cells or Jurkat cells;
(2) contacting an anti-leukemia chemotherapeutic agent with the pre-treated Reh cells or Jurkat cells obtained in step (1).
25. A method for screening a drug for treating or preventing drug-resistant relapse of acute lymphoblastic leukemia in children, which comprises the following steps:
(1) contacting a candidate compound with Reh cells or Jurkat cells comprising a PRPS1 mutant gene from any of the PRPS1 mutant gene populations of claim 8 or 9 to obtain preconditioned Reh cells or Jurkat cells;
(2) contacting an anti-leukemia chemotherapeutic agent with the pre-treated Reh cells or Jurkat cells obtained in step (1).
26. The method of claim 25, wherein said contacting comprises adding the candidate compound directly to the culture medium of said Reh cells or Jurkat cells; and/or, step (1) is also performed without simultaneously contacting the candidate compound with Reh cells or Jurkat cells containing any of the PRPS1 mutant genes in the PRPS1 mutant gene population of claim 8 or 9 as a control; and/or the chemotherapeutic drug is a purine drug; and/or the purine medicine is 6-MP or 6-TG.
27. The method of claim 25, further comprising the step of (3) detecting the viability of the cells obtained in step (2); and/or the detection method is a counting method after staining by using a living cell dye, an MTT method or a method for measuring the fluorescence intensity emitted after the luciferase is combined with ATP in the living cells; and/or, the over-measurement of the fluorescence intensity of luciferase after binding to ATP in the surviving cells is carried out by using a commercially available Cell Titer-Glo reagent from Promega corporation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510086559.5A CN105986023B (en) | 2015-02-17 | 2015-02-17 | Acute lymphocytic leukemia drug-resistant relapse related mutant gene and application thereof |
| PCT/CN2016/073906 WO2016131420A1 (en) | 2015-02-17 | 2016-02-17 | Mutant gene related to drug resistance and relapse of acute lymphoblastic leukaemia and use thereof |
| US15/551,583 US11021753B2 (en) | 2015-02-17 | 2016-02-18 | Mutant genes related to drug resistance and relapse of acute lymphoblastic leukaemia and a use thereof |
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