WO2016131420A1 - 急性淋巴细胞白血病耐药复发相关突变基因及其应用 - Google Patents

急性淋巴细胞白血病耐药复发相关突变基因及其应用 Download PDF

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WO2016131420A1
WO2016131420A1 PCT/CN2016/073906 CN2016073906W WO2016131420A1 WO 2016131420 A1 WO2016131420 A1 WO 2016131420A1 CN 2016073906 W CN2016073906 W CN 2016073906W WO 2016131420 A1 WO2016131420 A1 WO 2016131420A1
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prps1
replaced
mutant
cell
drug
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French (fr)
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李本尚
李慧
王升跃
周斌兵
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上海交通大学医学院附属上海儿童医学中心
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Priority claimed from CN201510086559.5A external-priority patent/CN105986023B/zh
Priority claimed from CN201510086558.0A external-priority patent/CN105985960B/zh
Application filed by 上海交通大学医学院附属上海儿童医学中心 filed Critical 上海交通大学医学院附属上海儿童医学中心
Priority to US15/551,583 priority Critical patent/US11021753B2/en
Publication of WO2016131420A1 publication Critical patent/WO2016131420A1/zh

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Definitions

  • the invention relates to the field of biomedicine, in particular to an acute lymphoblastic leukemia drug-resistant recurrence-related mutant gene and application thereof.
  • ALL Acute Lymphoblastic Leukemia
  • Chemotherapy is one of the main means of induction and post-remission treatment during the treatment of childhood ALL.
  • chemotherapy drugs commonly used in clinical practice usually use nucleotide analogs to destroy DNA and RNA synthesis, thereby causing DNA damage to induce tumor cell apoptosis.
  • 90% of patients will be relieved by combination chemotherapy with high-dose drugs.
  • the treatment will be maintained by low-dose chemotherapeutic drugs.
  • it is mainly through oral administration of 6-mercaptopurine (6).
  • -MP 6-ostrich (6-TG)
  • tumor cells need to tolerate 6-MP/6-TG to accumulate recurrence.
  • N5C2 5'-nucleotidase II
  • the gene is responsible for encoding the metabolic related enzymes of nucleotides and nucleotides similar to 6-mercaptopurine (6-MP) and 6-ostrich (6-TG), and the mutation ratio in the resistance relapse of ALL is about 10 %.
  • the drug-resistant recurrence of childhood ALL is not a single-base mutation of a gene.
  • the search for genetic mutations related to recurrence is still the focus and difficulty of the study of drug-resistance recurrence in children, and the corresponding detection and treatment methods More lacking.
  • the PRPS1 gene encodes a phosphoribosyl pyrophosphate synthase, the first rate-limiting enzyme in the intracellular indole metabolism synthesis pathway.
  • PRPS1 catalyzes the reaction of 5-phosphate ribose with ATP to form AMP and PRPP (5-phosphate ribose-1-pyrophosphate).
  • PRPP undergoes a series of enzyme-catalyzed reactions to produce hypoxanthine nucleotides (IMP), and IMP is further converted to (d) ATP.
  • IMP hypoxanthine nucleotides
  • IMP hypoxanthine nucleotides
  • IMP hypoxanthine nucleotides
  • raw materials for DNA and RNA synthesis such as GTP. Too high or too little intracellular PRPP may cause abnormalities in the metabolic pathways that use it as a substrate or regulator, leading to the occurrence of disease.
  • PRPS1 Mutations in PRPS1 have been found to be associated with disease, such as X-linked nonsyndromic sensorineural deafness (DFN2) and progressive neuropathic muscular atrophy syndrome (X-linked Charcot-Marie-Tooth disease- 5, CMTX5) and so on.
  • the PRPS1 mutation has a Gain of function, such as D183H, A190V, D52H, etc., and also has a reduced function such as A87T, M115T, I290T and the like. But so far no mutations have been found in PRPS1 in tumors. .
  • Lometrex is a folic acid antagonist (antifolate) which inhibits the activity of glycinate nucleotide formyltransferase (GART) by transporting a reducing folate carrier and a folate receptor into a cell.
  • GART is a de novo A key enzyme in the synthetic pathway.
  • the drug is also effective as a potential anti-tumor drug against some cell lines that are resistant to other anti-tumor drugs.
  • the drug has already completed Phase II clinical trials in foreign countries, but so far there has been no report on the drug's resistance to relapse in the prevention and treatment of acute lymphoblastic leukemia.
  • the technical problem to be solved by the present invention is to provide a group of acute lymphoblastic leukemia drug-resistant recurrence-related mutation gene groups and to evaluate the resistance of children ALL in the prevention of relapse and detection of drug resistance in childhood ALL.
  • the application of drug recurrence risk is to provide a group of acute lymphoblastic leukemia drug-resistant recurrence-related mutation gene groups and to evaluate the resistance of children ALL in the prevention of relapse and detection of drug resistance in childhood ALL. The application of drug recurrence risk.
  • the present invention also provides a target for preventing or treating drug-resistant relapse of acute lymphoblastic leukemia, an RNA drug for treating acute leukemia drug-resistant recurrence, and an inhibitor of one or more enzymes of all enzymes in the de novo synthesis pathway in preparation
  • the invention has important guiding significance for gene diagnosis and individualized treatment in the early stage of clinical recurrence.
  • PRPS1 is used as a genetic marker for drug-resistant recurrence of acute lymphoblastic leukemia way.
  • the present invention also provides a PRPS1 mutant, wherein the amino acid sequence of the PRPS1 mutant is formed by a substitution mutation of an amino acid at the following position in the sequence shown by SEQ ID No. 2 in the Sequence Listing, the substitution being: The 103th serine was replaced by threonine, the 103rd serine was replaced by asparagine, the 144th asparagine was replaced by serine, the 176th lysine was replaced by asparagine, and the 183th aspartic acid was replaced.
  • the 190th alanine is replaced by threonine
  • the 191th leucine is replaced by phenylalanine
  • the 303th threonine is replaced by serine
  • the 53rd valine is replaced by alanine.
  • the 72th isoleucine is replaced by proline
  • the 77th cysteine is replaced by serine
  • the 139th aspartic acid is replaced by glycine
  • the 311th tyrosine is replaced by cysteine
  • the 103th serine was replaced with isoleucine
  • the 114th asparagine was replaced with aspartic acid or the 174th glycine was replaced with glutamic acid.
  • the mutant in which the serine at position 103 is replaced with threonine is represented as S103T in the present invention, and the expression patterns of other mutants are the same, followed by S103N, N144S, K176N, L191F, D183E, A190T, T303S, V53A. , I72V, C77S, D139G, Y311C, S103I, N114D or G174E.
  • the present invention provides a PRPS1 mutant gene encoding a PRPS1 mutant of the above amino acid sequence.
  • the PRPS1 mutant gene may be as conventional in the art, and may be a PRPS1 mutant capable of encoding the above amino acid sequence due to the degeneracy of the base.
  • the DNA sequence of the mutated gene is preferably formed by a substitution mutation of a nucleotide at a position as shown in SEQ ID No. 1 of the Sequence Listing, which is replaced by the substitution of G at position 308 to C.
  • the 308th G is replaced by A
  • the 431st A is replaced by G
  • the 528th G is replaced by C
  • the 549th C is replaced by G
  • the 568th G is replaced by A
  • the 573th G is replaced by C
  • 908 bit C is replaced by G
  • 158th bit T is replaced by C
  • 214th bit A is replaced by G
  • 230th bit is replaced by C
  • 416th bit is replaced by G
  • 932th bit is replaced by G
  • 308th place G is replaced by T
  • 340th A is replaced by G or 521th G is replaced by A.
  • G308C The replacement of C at position 308 with C is represented by G308C in the present invention, and other substitutions are similar to G308A, A431G, G528C, C549G, G568A, G573C, C908G, T158C, A214G, G230C, A416G, A932G, G308T, respectively. , A340G or G521A.
  • the present invention provides a recombinant vector comprising the PRPS1 mutant gene of the present invention.
  • the recombinant vector is conventional in the art, preferably a prokaryotic expression vector or a eukaryotic expression vector capable of expressing the PRPS1 mutant gene, more preferably a lentiviral expression vector capable of expressing the PRPS1 mutant gene, optimally It is a GV303 lentiviral expression vector (Jikai gene).
  • the preparation method of the expression vector conventionally true art is preferably prepared by the following method: The lentiviral vector cut with the restriction enzymes linearized by clontech's In-Fusion TM PCR Cloning Kit The kit exchanges the product obtained by PCR amplification of the PRPS1 mutant gene into the linearized lentiviral vector, and the recombinant is amplified by E. coli TOP10 to form a recombinant vector containing the PRPS1 mutant gene of the present invention.
  • 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 spontaneously, and the carried PRPS1 mutant gene of the present invention can be efficiently expressed, preferably a prokaryotic cell expressing the PRPS1 mutant gene of the present invention.
  • a eukaryotic cell more preferably a eukaryotic cell expressing the PRPS1 mutant gene of the present invention, and most preferably a Reh cell line expressing the PRPS1 mutant gene of the present invention
  • the preparation method of the transformant is conventional in the art, preferably For ligation using the lentiviral vector expressing the PRPS1 mutant gene of the present invention into a lentivirus and then infecting the eukaryotic cell, preferably using a lentiviral vector GV303 (Jikai gene) expressing the PRPS1 mutant gene of the present invention and virus packaging
  • the helper plasmid co-transfected HEK293T cell line was packaged into lentivirus and infected with eukaryotic cell Reh, and then sorted by green fluorescent positive cells by flow cytometry.
  • a second aspect of the present invention is a group of PRPS1 mutants for assessing the risk of relapse of acute lymphoblastic leukemia, the PRPS1 mutant population comprising the following PRPS1 mutant: the amino acid sequence thereof is shown in SEQ ID No. 2 of the Sequence Listing.
  • the 103th serine is replaced by threonine
  • the 103th serine is replaced by asparagine
  • the 144th asparagine is replaced by serine
  • the 176th lysine is replaced by asparagine
  • the 183th is ascending Replacement of glutamic acid with glutamic acid, substitution of alanine at position 190 with threonine, substitution of leucine at position 191 with phenylalanine or substitution of threonine at position 303 with serine, ie S103T, S103N, N144S, K176N, L191F, D183E, A190T or T303S.
  • the mutant population preferably further comprises one or more of the following PRPS1 mutants: the amino acid sequence thereof is replaced with a proline at position 53 in the sequence shown by SEQ ID No. 2 in the sequence listing.
  • Acid the 72th isoleucine is replaced by proline, the 77th cysteine is replaced by serine, the 139th aspartic acid is replaced by glycine, the 311th tyrosine is replaced by cysteine,
  • the 103th serine was replaced with isoleucine, the 114th asparagine was replaced with aspartic acid, the 174th glycine was replaced with glutamic acid or the 190th glycine was replaced with valine. That is, V53A, I72V, C77S, D139G, Y311C, S103I, N114D, G174E or A190V.
  • the present invention provides a group of PRPS1 mutant gene genes for assessing the risk of relapse of acute lymphoblastic leukemia, the PRPS1 mutant gene group comprising the following PRPS1 mutant gene: the nucleotide sequence thereof is as shown in SEQ ID No. 1 of the Sequence Listing.
  • the 308th G is replaced by C
  • the 308th G is replaced by A
  • the 431st A is replaced by G
  • the 528th G is replaced by C
  • the 549th C is replaced by G
  • the 568th G is replaced by A.
  • the 573th G is replaced by C or the 908th C is replaced by G. That is, G308C, G308A, A431G, G528C, C549G, G568A, G573C or C908G.
  • the mutant gene group preferably further comprises one or more of the following PRPS1 mutant genes: the nucleotide sequence thereof is replaced by C at position 158 of the sequence shown by SEQ ID No. 1 in the sequence listing.
  • Bit 214 A is replaced by G
  • 230th G is replaced by C
  • 416th A is replaced by G
  • 932th A is replaced by G
  • 308th G is replaced by T
  • 340th A is replaced by G
  • 521th Bit G is replaced by A or 569th C is replaced by T. That is T158C, A214G, G230C, A416G, A932G, G308T, A340G, G521A or C569T.
  • the present invention provides a kit for assessing the risk of relapse of ALL resistance, the kit comprising reagents and instructions for detecting the PRPS1 mutant gene in the mutated gene population of the present invention.
  • the reagent may be any reagent conventionally used in the art for detecting a PRPS1 mutant gene, and preferably includes one or more of a primer, a DNA polymerase, a dNTP or a buffer that amplifies each exon of the PRPS1 gene.
  • the primer for amplifying each exon of the PRPS1 gene is a primer which can amplify each exon of the PRPS1 gene, and preferably has a nucleotide sequence such as SEQ ID NO. 3 to SEQ ID NO in the sequence listing. Shown in any of .16.
  • the detection kit further comprises an agent for extracting DNA of a cell or tissue sample
  • the reagent for extracting DNA of the cell or tissue sample is conventional in the art, preferably a protease, a saturated phenol, and a volume ratio of 24:1.
  • the DNA polymerase is conventional in the art, preferably a KOD-Plus DNA polymerase produced by TOYOBO Corporation.
  • the dNTPs are conventional in the art, preferably a mixture of four of dATP, dGTP, dCTP and dTTP.
  • the buffer is a buffer system compatible with the DNA polymerase, preferably a KOD-Plus DNA polymerase buffer produced by TOYOBO.
  • the instruction manual describes the steps of using the kit, and the contents described in the preferred instruction manual include the following steps:
  • 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 the PCR-amplified PRPS1 gene is obtained by using the sample genomic DNA obtained in the step (1) as a template. For each exon, the amplified fragment was sequenced.
  • the instructions for use preferably further comprise the step (3): detecting the presence of any one or more of the PRPS1 mutant genes in the genomic DNA of the sample, the sample has acute lymphoblastic leukemia resistance The risk of recurrence. If the PRPS1 mutant gene is present, the sample is considered to be at risk of drug relapse.
  • the present invention provides a method for assessing the risk of drug resistance recurrence in a patient's ALL, comprising the following steps:
  • the sample is conventional in the art, preferably a bone marrow-derived or peripheral blood-derived tumor cell or an in vitro cultured immortalized cell line of the patient to be examined.
  • the extracted sample genomic DNA is conventional in the art, preferably extracted using a DNA extraction kit manufactured by Qiagen.
  • 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 comprises: PCR amplification using the sample genomic DNA obtained in the step (1) as a template Each exon of the PRPS1 gene was sequenced.
  • the PCR amplification is routine in the art and is preferably amplified using the following conditions:
  • Genomic DNA template 1 ⁇ l (50ng)
  • the sequencing is routine in the art, preferably one generation sequencing or second generation sequencing.
  • the detecting method further comprises the step (3) of assessing the risk of drug-resistant relapse of acute lymphoblastic leukemia according to the following criteria: detecting the presence of any one or more of the PRPS1 mutant gene group in the sample genomic DNA For the PRPS1 mutant gene, the sample has a risk of relapse of acute lymphoblastic leukemia.
  • the third technical solution of the present invention a kit for evaluating the risk of drug relapse of acute lymphoblastic leukemia, comprising: an agent for detecting a PRPS1 mutant in the PRPS1 mutant population and an instruction manual.
  • Such reagents are routine in the art, preferably reagents for detecting the activity of the PRPS1 mutant enzyme, more preferably glucose and PRPP labeled with isotopic carbon atoms.
  • the isotope carbon atom-labeled glucose is conventional in the art, preferably a glucose labeled with the isotope 13 C, which is conventional in the art, preferably a PRPP available from Sigma.
  • the kit preferably further comprises a cell lysis reagent, which is conventional in the art, preferably 80% methanol, the percentage being a volume percent.
  • the kit preferably further comprises a glucose-free medium, which is conventional in the art, preferably a glucose-free medium available from Gibco.
  • the instructions for use describe the instructions for use of the kit, and the contents described preferably include the following steps:
  • the lysing of the sample cells described in the step (1) is conventional in the art.
  • the method of detecting the PRPS1 mutant in the cell lysate obtained in the step (1) in the step (2) is conventional in the art, and preferably the enzyme activity of the PRPS1 is detected.
  • the content described in the instruction manual preferably further comprises the step (3): detecting the presence of the PRPS1 mutant having higher enzymatic activity in the sample than the wild type PRPS1, the sample has a risk of relapse of acute lymphoblastic leukemia resistance.
  • the PRPS1 mutant is preferably any one or more of the PRPS1 mutant populations described in the second aspect of the present invention.
  • the present invention provides a method for assessing the risk of relapse of childhood ALL resistance by detecting the activity of PRPS1 enzyme, the method comprising the steps of:
  • the method comprises the following steps:
  • step (b) cultivating the leukemia cells described in step (a);
  • step (c) treating the leukemia cells of step (b) with a chemotherapeutic drug
  • step (d) collecting the leukemia cells of step (c), lysing
  • the chemotherapeutic agent is conventional in the art, preferably a guanidine analog chemotherapeutic drug, more preferably 6-MP or 6-TG.
  • step (d) the cleavage is conventional in the art, preferably using 80% methanol cracking.
  • step (e) the assay is routine in the art, preferably using a LC-MS to detect the PRPP content.
  • the fourth technical scheme of the present invention a method for detecting the drug activity for treating or preventing drug resistance recurrence in children, comprising the following steps:
  • the method preferably further comprises the step (3), the step (3) comprising: detecting the viability of the cells obtained in the step (2).
  • the present invention provides a method for screening for a drug for treating or preventing drug-resistant relapse of childhood acute lymphoblastic leukemia, comprising the steps of:
  • the leukemia cell expressing the PRPS1 mutant gene of the present invention as described in the step (1) is conventional in the art, preferably a human cell, more preferably a human lymphocyte, and most preferably a Reh cell or a Jurkat cell.
  • the candidate compound is conventional in the art, preferably an existing compound, a natural product or a nucleic acid drug, more preferably a compound, a natural product or a nucleic acid drug that targets the purine synthesis pathway; the candidate compound has A drug that potentially reverses the effects of resistance to relapse in ALL.
  • the contacting is conventional in the art, preferably such that the candidate compound is capable of directly contacting the leukemia cell, more preferably the candidate compound is added directly to the culture medium of the leukemia cell.
  • the treatment in the step (2) is conventional in the art, preferably, the chemotherapeutic drug is capable of directly contacting the leukemia cell, and more preferably, the chemotherapeutic drug is directly added to the leukemia cell.
  • the chemotherapeutic drug is preferably a steroid drug, and the steroid drug is preferably 6-MP or 6-TG.
  • the candidate compound is not simultaneously contacted with leukemia cells containing the PRPS1 mutant gene of any one of the PRPS1 mutant gene groups according to claim 9 or 10 as a control.
  • the step (3) of detecting the viability of the cells obtained in the step (2) is preferred to further comprise the step (3) of detecting the viability of the cells obtained in the step (2).
  • the method for detecting in the step (3) is conventional in the art, and is preferably a method of staining after staining with a living cell dye, an MTT method, or a method of measuring fluorescence intensity after binding of luciferase to ATP in a living cell, Goodlands are tested using commercially available commercial assay kits such as Promega's Cell Titer-Glo reagent.
  • step (3) simultaneously detects the viability of the cells in the control in step (2). Comparing the survival rates, if the survival rate of the test cells is significantly lower than that of the control cells, it can be considered that the drug to be tested has an activity of reversing the resistance relapse of ALL.
  • the "resistance relapse” refers to the resistance of a patient with acute lymphoblastic leukemia to the treatment of a chemotherapeutic drug during the recovery period, which is conventional in the art, preferably a steroid chemotherapy drug, and more
  • a chemotherapeutic drug which is conventional in the art, preferably a steroid chemotherapy drug, and more
  • the preferred site is 6-mercaptopurine (6-MP) or 6-ostrich (6-TG).
  • prevention refers to preventing or reducing the drug resistance complex after use in the case where the drug resistance recurrence may exist. The occurrence of hair.
  • the “treatment” refers to alleviating the degree of relapse of the drug resistance, or curing the drug relapse to restore the normal lymphoblastic leukemia patient, or slow down the progression of the drug relapse.
  • a target for preventing or treating a drug resistant relapse of acute lymphoblastic leukemia wherein the target is one or more of all enzymes in the de novo synthesis pathway.
  • the target is preferably one or more selected from the group consisting of PRPS1, PRPS2, PPAT, PFAS, GART, ATIC, PAICS, and ADSL in the de novo synthesis pathway.
  • the sixth aspect of the present invention is a siRNA comprising a sense RNA fragment and an antisense RNA fragment, the sense RNA fragment comprising the DNA-encoded RNA molecule of the target of claim 1, the sense RNA fragment and Antisense RNA fragments are capable of complementing to form a double-stranded RNA molecule.
  • the seventh aspect of the present invention is a guide RNA which targets the GART gene, and the nucleotide sequence thereof is shown in SEQ. ID No. 41.
  • the eighth aspect of the present invention is a lentiviral vector, which comprises the DNA sequence of the guide RNA and the Cas9 gene as described in the seventh aspect of the present invention.
  • the empty vector of the lentiviral vector is conventional in the art, preferably an HIV-1 based lentiviral vector, more preferably a lenti CRISPR carrier purchased from addgene.
  • a ninth aspect of the present invention is a lentivirus which is packaged by a virus in a cell line with the aid of a lentiviral packaging plasmid as described in the eighth aspect of the present invention.
  • the lentiviral packaging plasmid is conventional in the art, preferably psPAX2 and pMD2.G, which are conventional in the art, preferably the HEK293T cell line.
  • the viral packaging is conventional in the art, preferably the lentiviral vector expressing the down-regulated GART gene is co-transferred with the lentiviral packaging plasmid into the cell line, and the culture medium is collected after culture. The supernatant was concentrated. The concentration is routine in the art, preferably by centrifugation of the collected virus solution through an Amicon Ultra-15100KD ultrafiltration tube (Millipore), centrifuged at 4 ° C for 30 minutes.
  • a guide RNA is targeted to the ATIC gene, and the nucleotide sequence thereof is shown in SEQ. ID No. 42.
  • the eleventh aspect of the present invention is a lentiviral vector, which comprises the DNA sequence of the guide RNA and the Cas9 gene as described in the tenth aspect of the present invention.
  • the empty vector of the lentiviral vector is conventional in the art, preferably an HIV-1 based lentiviral vector, more preferably a lenti CRISPR carrier purchased from addgene.
  • a lentivirus is provided by the lentivirus according to the eleventh technical solution of the present invention.
  • the body is packaged by virus in a cell line with the aid of a lentiviral packaging plasmid.
  • the lentiviral packaging plasmid is conventional in the art, preferably psPAX2 and pMD2.G, which are conventional in the art, preferably the HEK293T cell line.
  • the viral packaging is conventional in the art, preferably the lentiviral vector expressing the down-regulated ATIC gene is co-transferred with the lentiviral packaging plasmid into the cell line, and the culture medium is collected after culture. The supernatant was concentrated. The concentration is routine in the art, preferably by centrifugation of the collected virus solution through an Amicon Ultra-15100KD ultrafiltration tube (Millipore), centrifuged at 4 ° C for 30 minutes.
  • the Cas9 gene of the lentiviral vector is conventional in the art, preferably the Cas9 gene optimized according to the human genetic code.
  • a thirteenth aspect of the present invention a guide RNA according to the seventh and/or tenth aspect of the present invention, a lentiviral vector according to the eighth and/or eleventh aspects of the present invention, or the technique of the present invention
  • a pharmaceutical composition for treating or preventing relapse of drug resistance of acute lymphoblastic leukemia wherein the effective substance comprises siRNA selected from the sixth aspect of the present invention, and according to the seventh aspect of the present invention.
  • the effective substance comprises siRNA selected from the sixth aspect of the present invention, and according to the seventh aspect of the present invention.
  • the lentiviral vector according to the eighth and/or eleventh aspects of the present invention, and the lentivirus according to the ninth and/or twelveth aspect of the present invention are provided.
  • Fifteenth of the present invention is an application of an inhibitor of one or more enzymes of all enzymes in the de novo synthesis pathway in the preparation of a medicament for preventing or treating drug-resistant relapse of acute lymphoblastic leukemia.
  • the inhibitor is conventional in the art, preferably one or more of an inhibitory RNA, an inhibitory polypeptide, an antibody, and a small molecule compound inhibitor, more preferably an inhibitory RNA and a small molecule.
  • the inhibitory RNA is conventional in the art, preferably one or more selected from the group consisting of a guide RNA, siRNA, shRNA, miRNA, antisense RNA, and ribozyme, more preferably selected from the group consisting of a guide RNA, an siRNA, and One or more of the shRNAs, most preferably a guide RNA, such as a guide RNA as shown in SEQ ID NO. 41 or SEQ ID NO.
  • the small molecule compound inhibitor is conventional in the art, preferably one or two selected from the group consisting of Lometrexol and Alimta, more preferably Lometripa.
  • the "resistance relapse” refers to the resistance of a patient with acute lymphoblastic leukemia to the treatment of a chemotherapeutic drug during the recovery period, which is conventional in the art, preferably a steroid chemotherapy drug, and more
  • a chemotherapeutic drug which is conventional in the art, preferably a steroid chemotherapy drug, and more
  • the preferred site is 6-mercaptopurine (6-MP) or 6-ostrich (6-TG).
  • prevention refers to preventing or reducing the occurrence of the drug relapse after use in the case where the drug resistance recurrence may exist.
  • treating refers to reducing the degree of relapse of the drug resistance, or curing the drug relapse to cause acute lymphoid Patients with leukemia return to normal or slow the progression of the drug relapse.
  • the medicament for preventing or treating drug-resistant relapse of acute lymphoblastic leukemia includes one or more of the inhibitory RNA, an inhibitory polypeptide, an antibody, and a small molecule compound inhibitor, and a pharmaceutically acceptable carrier.
  • one or more of the inhibitory RNA, the inhibitory polypeptide, the antibody, and the small molecule compound inhibitor may be used as an active ingredient alone or together with other components.
  • the active ingredient means a function of preventing or treating the relapse of the drug resistance.
  • the pharmaceutically acceptable carrier includes pharmaceutically acceptable excipients, fillers, diluents and the like.
  • the content of one or more of the inhibitory RNA, the inhibitory polypeptide, the antibody, and the small molecule compound inhibitor is 0.01 to 99.99%, and the content of the pharmaceutically acceptable carrier is 0.01 to 99.99%.
  • the percentage is the mass percentage of the total mass of the drug.
  • the dosage form of the drug is not particularly limited and may be in the form of a solid, a semi-solid or a liquid, and may be an aqueous solution, a non-aqueous solution or a suspension, or may be a tablet, a capsule, a granule, an injection or an infusion. It can be administered orally, or it can be administered by intravenous, intramuscular, intradermal or subcutaneous injection.
  • the dosage of the drug for preventing or treating acute lymphoblastic leukemia drug relapse is determined according to the age and condition of the patient, and the usual daily dose is about 0.0001 to 1000 mg/kg body weight, preferably It is 0.01 to 500 mg/kg body weight, more preferably 0.1 to 200 mg/kg body weight.
  • the number of administrations is once or several times a day.
  • the drug for preventing or treating drug-resistant relapse of acute lymphoblastic leukemia can also be used in combination with a chemotherapeutic drug at the time of treatment.
  • the chemotherapeutic agent is conventional in the art, preferably a steroid chemotherapeutic drug, more preferably 6-mercaptopurine (6-MP) or 6-ostrich guanidine (6-TG).
  • a sixteenth technical solution of the present invention a kit for assessing the risk of relapse of acute lymphoblastic leukemia, which reagent for lysing sample cells, detecting hypoxanthine (HX), hypoxanthine nucleotide monophosphate (IMP), Reagents and instructions for use of one or more of 5-aminoimidazole-4-carboxamide nucleotides (AICAR) and inosine (Inosine); the contents described in the instructions for use include the following steps:
  • step (c) determining the acute lymphocyte of the sample by the concentration of one or more of hypoxanthine, hypoxanthine nucleotide monophosphate, 5-aminoimidazole-4-carboxamide nucleotide and inosine obtained in step (b) The risk of relapse of leukemia resistance.
  • the kit preferably further comprises a standard, which is conventional in the art, preferably in the form of hypoxanthine, 5-aminoimidazole-4-carboxamide nucleotide and inosine.
  • a standard which is conventional in the art, preferably in the form of hypoxanthine, 5-aminoimidazole-4-carboxamide nucleotide and inosine.
  • a standard which is conventional in the art, preferably in the form of hypoxanthine, 5-aminoimidazole-4-carboxamide nucleotide and inosine.
  • the reagent for lysing the sample cells is conventional in the art, preferably 80% methanol, and the percentage is a volume percentage.
  • the reagent for detecting one or more of hypoxanthine, hypoxanthine nucleotide monophosphate, 5-aminoimidazole-4-carboxamide nucleotide and inosine is conventional in the art, preferably.
  • the ground is a mass spectrometric detection reagent.
  • the invention also provides a method for assessing the risk of relapse of acute lymphoblastic leukemia, the method comprising the steps of:
  • step (c) determining the acute lymphocyte of the sample by the concentration of one or more of hypoxanthine, hypoxanthine nucleotide monophosphate, 5-aminoimidazole-4-carboxamide nucleotide and inosine obtained in step (b) The risk of relapse of leukemia resistance.
  • the sample cells in the step (a) may be an acute lymphoblastic leukemia sample cell which is conventional in the art, preferably an acute lymphoblastic leukemia sample cell containing the PRPS1 mutant as described above, more preferably Reh cells comprising a PRPS1 mutant as described above, most preferably the aforementioned Reh-PRPS1-S103T or Reh-PRPS1-A190T cells; the cleavage can be carried out by methods conventional in the art, preferably using 80% methanol
  • the sample cells are lysed, the percentage being a volume percent; in step (b), the detection can be a conventional detection method in the art, preferably mass spectrometry.
  • the reagents and starting materials used in the present invention are commercially available.
  • the positive progressive effect of the present invention is that the present invention provides a mutated gene associated with drug-resistance recurrence of acute lymphoblastic leukemia in the treatment of drug-resistant relapse in the treatment of acute lymphoblastic leukemia with a nucleoside analog as a chemotherapeutic drug. It provides a new target for the treatment of drug-resistant relapse of ALL.
  • the kit for assessing the risk of relapse of ALL based on the mutated gene can realize the risk assessment of drug-resistant relapse of childhood acute lymphoblastic leukemia.
  • the present invention provides a novel drug for the treatment of acute lymphoblastic leukemia in the treatment of acute lymphoblastic leukemia with the use of a purine analog as a chemotherapeutic drug, and provides a novel therapeutic agent for the action of the enzyme in the purine synthesis pathway.
  • the application of preventing relapse of drug resistance in acute lymphoblastic leukemia also provides a novel kit for detecting the risk of drug-resistant recurrence of acute lymphoblastic leukemia, which detects metabolite hypoxanthine nucleotides, hypoxanthine, AICAR and Inosine selected from the purine synthesis pathway.
  • One or more of them as an indicator to assess the risk of drug resistance recurrence in ALL, provides a powerful technical means and support for the prevention and treatment of drug resistance recurrence in children.
  • Figure 1 Ultra-deep sequencing revealed that PRPS1 is a relapse-specific mutation and the proportion of mutations increases rapidly before clinical relapse.
  • FIG. 1 Schematic diagram of the structure of PRPS1 mutant protein.
  • Figure 3 Map of the pET28a vector.
  • Figure 4 Results of SDS-PAGE electrophoresis of purified PRPS1 wild type and each mutant.
  • Figure 5 PCR amplification of PRPS1 wild-type agarose gel electrophoresis results.
  • Figure 6 Western results of stable Reh cell line expressing PRPS1 wild type and each mutant.
  • Figure 7 Drug sensitivity results for 6-MP and 6-TG of stable Reh cell lines expressing PRPS1 wild type and each mutant.
  • Figure 8 Results of apoptosis of 6-MP and 6-TG by stable Reh cell line expressing PRPS1 wild type and each mutant.
  • Figure 9 Metabolite detection results of the intracellular chemotherapeutic drug 6-MP/6-TG.
  • Figure 10 Detection results of PRPS1 enzyme activity expressing PRPS1 wild type and each mutant.
  • Figure 11 ADP and GDP for the PRPS1 feedback adjustment path.
  • Figure 12 Effect of GDP/ADP on inhibition of PRPS1 protein activity.
  • Figure 13 Results of determination of product content in the intracellular sputum metabolic pathway.
  • Figure 14 Inhibition of PRPS1S103T and A190T mutant resistance by a nucleic acid drug CRISPR primer RNA targeting the purine synthesis pathway.
  • Figure 15 Exogenously added sputum affects drug tolerance of Reh cells to the chemotherapeutic drug 6-MP.
  • Figure 16 Exogenously added sputum affects the metabolism of Reh cells to the chemotherapeutic drug 6-MP.
  • Figure 17 Hypoxanthine competitively inhibits the response of the chemotherapeutic drug 6-MP.
  • Figure 18 Lometrexol reverses 6-MP drug tolerance caused by mutations in the PRPS1 gene.
  • Figure 19 Inhibition of resistance of PRPS1S103T to A190T mutant by nucleic acid drug shRNA targeting the purine synthesis pathway.
  • the present invention uses a whole exome sequencing technique to sequence the initial, remission and recurrence samples of 16 groups of children with ALL, and finds that recurrence-specific mutations of multiple sites of the Phosphoribosyl pyrophosphate synthase I gene exist in the scorpion synthesis rate-limiting enzyme PRPS1 (Phosphoribosyl pyrophosphate synthase I) gene. It is wild type in both initial and remission samples. By further PRPS1 sequencing of 144 relapsed samples, it was found that mutations in the PRPS1 gene occurred in 16 relapsed samples, and the mutation frequency was 13% (18/138) in B-ALL. At the same time, the Charotti School of Medicine in Germany (Charotti- Dr.
  • the invention combines cell biology, molecular biology, metabolomics and the like to systematically study the PRPS1 gene mutation from the influence of PRPS1 gene mutation on its own enzyme activity and the influence of 6-MP drug metabolism and purine metabolism network. Guide the mechanism of drug resistance recurrence in children. The study reveals a new drug-resistance recurrence mechanism in childhood ALL, suggesting that PRPS1 mutation can drive recurrence, is a genetic marker for drug resistance and recurrence, and has important guiding significance for early clinical diagnosis and individualized treatment of recurrence. .
  • the D183H mutant involved in the following examples is a functionally acquired mutant of the reported PRPS1 gene; the A87T and M115T mutants involved are reported functional deletion mutants, which serve as assays for assays, respectively. Whether it is normal positive control and negative control.
  • the S103T, S103N, N144S, T303S, K176N, D183E, A190T, A190V and L191F involved are experimental groups. These mutants can be divided into two types by aligning the crystal structure of human PRPS1, that is, located at the interface of PRPS1 dimer formation.
  • the mutation site is located at the mutation site located at the PRPS1 allosteric site, as shown in Figure 2.
  • the mutant within the group is a resistant mutant of the PRPS1 gene.
  • PRPS1 was sequenced in 16 groups of children with primary, remission and recurrence and 144 relapsed samples by whole exome sequencing.
  • Quality control of the sample The quality of the sample is directly related to the reliability of the sequencing results.
  • the quality of the sample used for deep sequencing and subsequent verification is guaranteed by the following three methods: 1 Leukemia cells in the bone marrow may be present at the onset and recurrence of leukemia. More than 90% of nuclear cells can be removed by Ficoll density gradient centrifugation to remove residual normal granulocytes and nucleated red blood cells, so that high-purity leukemia cells can be obtained for subsequent DNA extraction and sequencing; The combination of light and antibody can distinguish leukemia cells from normal bone marrow hematopoietic cells. High purity (>99%) samples can be obtained by flow cytometry for samples with lower purity; 3, residual leukemia is selected according to the results of minimal residual disease detection. Samples with less than 0.01% of the remission phase are used for deep sequencing, thus ensuring the reliability of both initial and recurrent specific mutations.
  • Sample preparation The genomic DNA of leukemia cells was extracted by QIAGEN Blood DNA kit, the DNA concentration was determined by Q-bit fluorescence quantification kit, and high-purity genomic DNA was used for later deep sequencing.
  • Amplification of each exon of PRPS1 The appropriate primers were designed for the PRPS1 genomic sequence to amplify the exons of PRPS1.
  • the PCR system and program are as follows:
  • Genomic DNA template 1 ⁇ l (50ng)
  • the primer sequences are shown in Table 1.
  • the PCR products were subjected to capillary electrophoresis sequencing and mutation analysis as usual, and the mutation analysis software was Mutation Surveyor.
  • PRPS1 There was a recurrence-specific mutation in PRPS1 in German children ALL, with a mutation rate of 2.7% (6/220) (see Table 2). Combined with clinical pathology analysis, patients with mutations in the PRPS1 gene had early recurrence (P ⁇ 0.005) (see Table 3). Ultra-deep sequencing revealed that PRPS1 is a relapse-specific mutation and the proportion of mutations increases rapidly before clinical relapse. This means that PRPS1 drives the patient's drug-resistant recurrence, which can be used as a genetic marker for clinical recurrence diagnosis and an important target for clinical recurrence therapy.
  • the whole exosome sequencing technique was used to sequence the bone marrow samples of children with primary onset, remission and recurrence.
  • the mutations of PRPS1 gene were found in 18 patients with recurrence samples, and the mutation frequency was 13% (18/138), as shown in Table 2. Show. Among them, 9 of the relapsed samples were site mutations of A190T. Through the Charotti School in Germany (Charotti- Dr. Renate Kirschner-Schwarb of the Berlin Children's Hematology Center also confirmed the presence of recurrent specific mutations in PRPS1 in German children ALL with a mutation rate of 2.7% (6/220).
  • the 1 P value was calculated using Fisher's exact test.
  • Recurrence time earlier, within 18 months after diagnosis; early, between 18 months and 36 months after diagnosis; late, 36 months after initial treatment.
  • PRPS1 mutations occurred in patients with early recurrence (Chinese patients, P ⁇ 0.005, German patients, P ⁇ 0.001), indicating a relapsing diagnosis of recurrence.
  • PCR primer sequence of wild type PRPS1 was designed according to the sequence of PRPS1 (gene ID: 5631, NM 002764) provided by NCBI, forward primer: 5' cgcggcagccatATGCCGAATATCAAAATCTTCAG 3', reverse primer: 5'gtggtggtgctcgagTTATAAAGGGACATGGCTGAATAGGTA3' (primer description: containing exchange pairing base, restriction site, and containing the 5' partial sequence of the gene of interest for PCR fishing for the gene of interest).
  • PRPS1 gene ID: 5631, NM 002764
  • forward primer 5' cgcggcagccatATGCCGAATATCAAAATCTTCAG 3'
  • reverse primer 5'gtggtggtgctcgagTTATAAAGGGACATGGCTGAATAGGTA3' (primer description: containing exchange pairing base, restriction site, and containing the 5' partial sequence of the gene of interest for PCR fishing for the gene of interest).
  • the recombinant plasmid clontech company by In-Fusion TM PCR Cloning Kit Kit PCR product from Step 1 exchanged into the linearized viral vector, TOP10 E. coli by amplification of recombinants.
  • PRPS1 Mutant Prokaryotic Vector Using pET28a-PRPS1 as a plasmid template, 9 mutants of PRPS1 were constructed by circular PCR of TOYOBO KOD-Plus DNA polymerase: S103T, S103N, N144S, T303S, K176N, D183E, A190T, A190V and L191F, PCR primers are shown in Table 5.
  • the PCR product was digested with 1 ⁇ l of DpnI enzyme in a 37 ° C water bath for 1 hour to digest the plasmid template, and 10 ⁇ l of the solution was added to E. coli TOP10 for amplification. The transformants were sequenced to verify whether the mutants were successfully constructed.
  • the bacteria were inoculated at 1:100 on the 2nd day, that is, 50 ⁇ l of the bacterial liquid was taken into 5 ml of LB (containing antibiotics), and cultured at 37 ° C, shaking at 220 rpm for 2 to 3 hours until the OD value reached 0.6.
  • Control sampling Take 2ml of bacterial solution, centrifuge at 12000rpm for 1 minute, discard the supernatant, add 1ml PBS solution to the bacterial pellet, sonicate (200w, ultrasonic for 3 seconds, intermittent 5 seconds, 20 cycles), add SDS-PAGE Buffer (reduced, 4x), 100 ° C, water bath for 10 minutes, centrifuged at 12000 rpm for 10 minutes, the supernatant was taken and stored at -20 ° C as a pre-induction control.
  • the inducing agent IPTG was added to a final concentration of 1 mM, cultured at 16 ° C, shaking at 220 rpm for 16 hours, centrifuged at 12,000 rpm for 1 minute, the supernatant was discarded, sonicated and sampled, and stored at -20 ° C.
  • PRPS1 is widely expressed:
  • Control sampling Take 2ml of bacterial solution, centrifuge at 12000rpm for 1 minute, discard the supernatant, add 1ml PBS solution to the bacterial pellet, sonicate (200w, ultrasonic for 3 seconds, intermittent 5 seconds, 20 cycles), add SDS-PAGE Buffer (reduced, 4x), 100 ° C, water bath for 10 minutes, centrifuged at 12000 rpm for 10 minutes, the supernatant was taken and stored at -20 ° C as a pre-induction control.
  • PRPS1 protein Purify the bacterial cells by ultrasonication, purify the series of PRPS1 proteins through the nickel column of AKTA-purifier system, and identify the expression and purity of PRPS1 series proteins by SDS-PAGE electrophoresis, and pass the BCA protein of Biyuntian Company.
  • the PRPS1 series protein was quantified by a quantitative kit to obtain an amino acid sequence such as the wild type PRPS1 protein shown by SEQ ID No. 2 in the Sequence Listing, as shown in FIG.
  • the PCR primer sequence of wild type PRPS1 was designed according to the sequence of PRPS1 (gene ID: 5631, NM 002764) provided by NCBI. Forward primer: 5'GAGGATCCCCGGGTACCGGTCGCCACCATGCCGAATATCAAAATC 3', reverse primer: 5'TCCTTGTAGTCCATACCGTGGTGGTGGTGGTGGTGCTCGAGTAAAG3 ', the primer contains an exchange paired base, a restriction site, and contains a 5'-end sequence of the target gene for PCR fishing for the gene of interest.
  • PCR was used to capture PRPS1, and the PCR product size was 1022 bp, as shown in FIG.
  • the PCR product was digested with 1 ⁇ l of DpnI enzyme in a 37 ° C water bath for 1 hour to digest the plasmid template, and 10 ⁇ l of the solution was added to E. coli TOP10 for amplification. The transformants were sequenced to verify whether the mutants were successfully constructed.
  • the recombinant plasmid clontech company by In-Fusion TM PCR Cloning Kit according to kit instructions PCR product from Step 1 exchanged into the linearized viral vector, TOP10 GV303-PRPS1 amplified by recombinant E. coli.
  • PRPS1 Mutant Plasmid Using the GV303-PRPS1 as a plasmid template, 9 mutant lentiviral expression vectors of PRPS1 were constructed by circular PCR of TOYOBO KOD-Plus DNA polymerase: 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, PCR Primers are shown in Table 5.
  • HEK293T cells were seeded into 10 cm dishes 24 hours before transfection, and transfection was started when the cell density reached 50% to 80% the next day.
  • the collected virus supernatant was added to an Amicon Ultra-15100KD ultrafiltration tube and centrifuged at 4 ° C for 30 minutes to obtain a concentrated virus solution.
  • Cell seeding Reh cells were counted and seeded in a 12-well plate, and 3 ⁇ 10 5 cells were seeded per well.
  • Flow sorting cells 72 hours after virus infection, the cells were sorted by green cell fluorescence Reh cells by Beckman's flow cytometer Moflo XDP, and the cells were expanded.
  • 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-PRPS1-L191F.
  • a stable cell line Reh-PRPS1-WT stably overexpressing wild-type PRPS1 was constructed using the GV303-PRPS1 lentiviral expression vector in the same manner as described above.
  • Reh cells are a human acute B lymphocyte leukemia strain.
  • steps are as follows:
  • the drug 6-MP was serially diluted, the initial concentration was 100 ⁇ g / ml, diluted 3 times in sequence, diluted 10 gradients, added to the well-packed 96-well plate, cultured at 37 ° C for 72 hours;
  • the sensitivity of different PRPS1 mutants to the chemotherapeutic drug 6-TG was determined by detecting the survival rate of Reh cells expressing different PRPS1 mutant genes after treatment with the chemotherapeutic drug 6-TG. The steps are as follows:
  • the drug 6-TG was serially diluted, the initial concentration was 100 ⁇ g / ml, diluted 3 times in sequence, diluted 10 gradients, added to the well-packed 96-well plate, cultured at 37 ° C for 72 hours;
  • Reh cells expressing wild type and various mutants of PRPS1 were counted, inoculated into 12-well plates, and each well was inoculated with 3 ⁇ 10 5 cells, and 2 replicate wells were set;
  • the metabolite of the intracellular chemotherapeutic drug 6-MP/6-TG was detected.
  • 6-MP is a prodrug, and it needs to be metabolized in vivo to form TIMP and TGMP before it functions, as shown in Figure 9C.
  • the detection steps are as follows:
  • Reh cells expressing PRPS1 wild type and various mutants were counted into 6 cm culture dishes, each dish was 3 ⁇ 10 6 cells;
  • 4LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) was used to detect the metabolites of chemotherapeutic drug 6-MP, TIMP, TGMP, r-MP, r-TG, r-MMP, respectively, using TIMP (Jean Bioscience, cat#NU- 1148), TGMP (Jean Bioscience, cat#NU-1121), r-MP (Sigma, cat#852686), r-TG (Sigma, cat#858412), r-MMP (Sigma, cat#M4002) as a standard curve Quantitative.
  • Experimental Results Figure 9 shows the intracellular content and alignment of 6-MP metabolites TIMP, TGMP and r-MP, r-TG, r-MMP for each mutant after 6-MP treatment in the experimental group. The ratio is significantly reduced.
  • the present invention combines the techniques of cell biology and metabolomics to detect the activity of PRPS1 in cells by means of isotope labeling.
  • the specific implementation is as follows:
  • Reh cells expressing PRPS1 wild type and various mutants were counted in a 6 cm culture dish, 3 ⁇ 10 6 cells per dish;
  • ATP (Cat. No. A7699)
  • ADP (Cat. No. A2754)
  • GDP (Cat. No. G7172)
  • Ribose-5-phosphate (Cat. No. R7750) were purchased from Sigma.
  • the reaction system is: 50mM Tris PH7.5, 2 mM phosphate, 1 mM DTT, 10 mM MgCl2, 0.5 mM ribose-5-phosphate, 0.5 mM ATP, PRPS1 protein.
  • the present invention combines the techniques of cell biology and metabolomics to detect the activity of PRPS1 in cells by means of isotope labeling, and on the basis of this, evaluates the effect of nucleotide GDP/ADP on PRPS1 activity.
  • the specific implementation is as follows:
  • Reh cells expressing PRPS1 wild type and various mutants were counted in a 6 cm culture dish, 3 ⁇ 10 6 cells per dish;
  • the cells were collected: the cells were transferred to a centrifuge tube, centrifuged at 3,000 g for 5 minutes, the supernatant discarded, in accordance with 3 ⁇ 10 6 cells / 200 ⁇ l 80% methanol, cells were lysed;
  • the content of 13 C 5 -PRPP was detected by 4LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC), and the standard curve was quantified by PRPP (sigma, cat#P8296). The change in the activity of PRPS1 enzyme was determined based on the content of PRPP.
  • the experimental results are shown in Figure 12C.
  • the concentration of PRPP in the wild-type experimental group decreased after the addition of nucleotide GDP/ADP.
  • the concentration of the catalytic reaction product PRPP of each PRPS1 mutant gene in the experimental group was not affected by nucleotide GDP/ADP. Effect, it was confirmed that the activity of the PRPS1 mutant enzyme in the cells expressing the PRPS1 mutant gene of the experimental group was not regulated by the feedback of the nucleotide GDP/ADP.
  • the invention combines the techniques of cell biology and metabolomics to detect the activity of the de novo synthesis pathway and the salvage synthesis pathway in the cells by the isotope labeling method.
  • the specific implementation is as follows:
  • Reh cells expressing PRPS1 wild type and various mutants were counted in a 6 cm culture dish, 3 ⁇ 10 6 cells per dish;
  • the present invention combines the techniques of cell biology and metabolomics to detect the content of hypoxanthine in cells by LC-MS.
  • the specific implementation is as follows:
  • 6LC-MS (ABI 5500Qtrap coupled with Waters Acquity UPLC) was used to detect the content of hypoxanthine, AICAR and Inosine, respectively, using 13 C 5 , 15 N 4 - hypoxanthine (Cambridge isotope laboratories, cat #489522), AICAR (sigma, Cat#A9978) and Inosine (sigma, cat#I4125) do absolute quantification of the standard curve.
  • CRISPR-ATIC TGAATTCGGTCCGCTTCCGGA (SEQ ID NO. 40)
  • CRISPR-GART GCAGCCCGAGTACTTATAAT (SEQ ID NO. 39)
  • Constructed into the CRISPR lentiviral vector (Addgene, cat#49535) to obtain lentiviral vector lentiCRISPR-ATIC and lentiCRISPR-GART;
  • Reh cells were counted and plated into 6 cm culture dishes, 3 x 10 6 cells per dish;
  • hypoxanthine (HX) or hypoxanthine nucleotide (IMP) was added to each 6 cm culture dish for 1 hour, and then treated with 10 ⁇ M chemotherapeutic drug 6-MP for 4 hours;
  • the chemotherapeutic drug 6-MP is an analogue of hypoxanthine, and both 6-MP and hypoxanthine are substrates of HGPRT.
  • the present invention establishes an in vitro enzymatic reaction to determine the Km value of the reaction of hypoxanthine with 6-MP and HGPRT. Reflects the affinity of hypoxanthine with 6-MP and HGPRT.
  • the specific implementation is as follows:
  • hypoxanthine and 6-MP were diluted in concentration gradient, reacted at 37 ° C for 1 hour, and 80% methanol was added to terminate the reaction. The contents of IMP and TIMP were detected by LC-MS. The reaction curves were plotted and the Km values of hypoxanthine and 6-MP were calculated by Graphpad 5.0 software.
  • Fig. 17B The affinity of hypoxanthine and HGPRT is significantly higher than that of 6-MP and HGPRT; and when 100 ⁇ M 6-MP is used as the reaction substrate, TIMP is added with the increase of the concentration of hypoxanthine. The generation is significantly suppressed.
  • Lometrexol reverses 6-MP drug tolerance caused by mutation of PRPS1 gene
  • Lometestil is a small molecule inhibitor of GART.
  • the invention detects that lomeracetan can reverse the 6-MP drug tolerance caused by the mutation of the PRPS1 gene, and the specific steps are as follows:
  • Thermo Fisher-70 °C ultra-low temperature refrigerator USA
  • Eppendorf 5810R high speed large capacity cryogenic centrifuge Germany
  • IKA Vibrax VXR small shaker Germany
  • IKA Vortex oscillator Germany
  • KQ5200DA ultrasonic cleaner Karlshan
  • the cells were collected by 80% methanol, centrifuged at 4 degrees 12000 rpm for 5 minutes, and the supernatant was transferred to a new EP tube, and 20 ⁇ l was taken for LC-MS/MS analysis.
  • Mobile phase composition Mobile phase A: 50 mM ammonium bicarbonate (pH 9.5)
  • MRM multi-channel reaction monitoring
  • 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
  • MRM multi-channel reaction monitoring
  • shRNA lentiviral vector GV298 against ATIC, GART and PPAT was purchased from the Jikai gene, and the corresponding shRNA sequences correspond to the following DNA sequences: shATIC-1 AATCTCTATCCCTTTGTAA:, shATIC-2: TGGAATCCTAGCTCGTAAT, shGART-1: CCAGGAGTTTGACTTACAA, shGART-2: CTAACTGTTGTCATGGCAA, shPPAT-1: CTTCGTTGTTGAAACACTT, shPPAT-2: TGTCTAACTGTAGACAAAT, shControl: TTCTCCGAACGTGTCACGT.
  • the control lentivirus LV-shControl, lentivirus LV-shATIC1, LV-shATIC2, LV-shGART1, LV-shGART2, LV-shPPAT1 and LV-shPPAT2 will be packaged.
  • the cell lines Reh, Reh-PRPS1-WT, Reh-PRPS1-S103T and Reh-PRPS1-A190T were respectively infected to form a stable cell line.
  • control cell lines Reh-shControl, Reh-shATIC1, Reh-shATIC2, Reh-shGART1, Reh-shGART2 , Reh-shPPAT1, Reh-shPPAT2, Reh-WT-shControl, Reh-WT-shATIC1, Reh-WT-shATIC2, Reh-WT-shGART1, Reh-WT-shGART2, Reh-WT-shPPAT1, Reh-WT-shPPAT2 , Reh-S103T-shControl, Reh-S103T-shATIC1, Reh-S103T-shATIC2, Reh-S103T-shGART1, Reh-S103T-shGART2, Reh-S103T-shPPAT1, Reh-S103T-shPPAT1, Reh-S103T-shPPAT2 , Reh-S103T-shControl, Reh-S103T-s

Abstract

本发明公开了急性淋巴细胞白血病(ALL)耐药复发相关突变基因PRPS1及其应用。还公开了作用于嘌呤合成途径中酶类的物质、包括洛美曲沙以及靶向GART和ATIC的抑制剂在制备预防或治疗ALL耐药复发的药物中的应用，与评估ALL耐药复发风险的试剂盒及其使用方法。

Description

急性淋巴细胞白血病耐药复发相关突变基因及其应用
本申请要求申请日为2015年2月17日的中国专利申请CN201510086559.5和申请日为2015年2月17日的中国专利申请CN201510086558.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药领域，具体涉及一种急性淋巴细胞白血病耐药复发相关突变基因及其应用。
背景技术
儿童急性淋巴细胞白血病(Acute Lymphoblastic Leukemia,ALL)是儿童期常见的恶性血液系统肿瘤疾病，是我国儿童因病死亡的重要原因之一。尽管在过去几十年经过众多努力后，儿童ALL的治疗和预后已有显著改善，但仍有15％～20％的患儿会复发，一旦复发，治愈率不足40％，复发一直是造成儿童ALL治疗失败和死亡的首要因素。复发问题日益成为国内外儿童ALL研究的热点和焦点。
儿童ALL治疗过程中，化疗是诱导缓解和缓解后治疗的主要手段之一。目前临床上常用的化疗药物通常是利用核苷酸类似物破坏DNA、RNA的合成，从而导致DNA损伤诱导肿瘤细胞凋亡。儿童ALL诱导缓解阶段，通过大剂量药物的联合化疗，有90％的病人会得到缓解，缓解后会通过低剂量的化疗药物来维持治疗，在维持治疗期主要是通过口服6-巯基嘌呤(6-MP)或6-巯鸟嘌呤(6-TG)，肿瘤细胞需要耐受6-MP/6-TG的作用才会积累复发。最近Adolfo Ferrando和William L Carroll的研究小组利用二代测序技术对耐药复发的白血病患者的样本进行检测，发现复发的ALL细胞中的5’-核苷酸酶II(NT5C2)基因存在突变，该基因负责编码与核苷酸及核苷酸类似药物6-巯基嘌呤(6-MP)和6-巯鸟嘌呤(6-TG)的代谢相关酶，在ALL耐药复发中的突变比例约为10％。儿童ALL的耐药复发并非是一个基因的单碱基突变可以解释的，寻找复发相关的遗传学变异仍是目前儿童ALL耐药复发研究的重点和难点，与之相应的检测与治疗手段也因此比较缺乏。
PRPS1基因编码磷酸核糖焦磷酸合成酶，是细胞内嘌呤代谢合成途径的第一个限速酶。PRPS1催化5-磷酸核糖和ATP的反应，生成AMP和PRPP(5-磷酸核糖-1-焦磷酸)。PRPP经过一系列酶催化反应后生成次黄嘌呤核苷酸(IMP)，IMP进一步转变为(d)ATP 和(d)GTP等DNA和RNA合成的原料。细胞内PRPP的含量过高或者过少，都可能引起以它为底物或者调节物的代谢通路异常，导致疫病的发生。目前发现PRPS1的突变与疾病相关，如非综合症型遗传性感音神经性耳聋(X-linked nonsyndromic sensorineural deafness,DFN2)、进行性神经病性肌萎缩综合症(X-linked Charcot-Marie-Tooth disease-5,CMTX5)等。PRPS1突变有功能获得性突变(Gain of function)，如D183H、A190V、D52H等，也有功能缺失性突变(Reduced function)，如A87T、M115T、I290T等。但到目前为止在肿瘤中PRPS1还没有发现突变。。
洛美曲沙(Lometrexol)，CAS号106400-81-1分子式C21H25N5O6，分子量443.45，结构式如下所示：
Figure PCTCN2016073906-appb-000001
洛美曲沙是一种叶酸拮抗剂(antifolate)，其被还原性叶酸载体与叶酸受体运输进入细胞后可以抑制甘氨酰胺核苷酸甲酰基转移酶(GART)的活性，GART是嘌呤从头合成途径所中的关键酶。该药物作为一种潜在的抗肿瘤药物对针对其他抗肿瘤药物产生抗药性的一些细胞系也有效果。该药物目前在外国已经完成二期临床实验，但至今尚没有任何关于该药物在预防和治疗急性淋巴细胞白血病的耐药复发中的报道。
发明内容
本发明所解决的技术问题：针对现在儿童ALL的耐药复发检测与治疗手段的较为缺乏的问题，本发明提供了一组急性淋巴细胞白血病耐药复发相关突变基因群及其在评估儿童ALL耐药复发风险中的应用。本发明还提供了预防或治疗急性淋巴细胞白血病耐药复发的药物的靶标、治疗急性白血病耐药复发的RNA药物和嘌呤从头合成途径中所有酶中的一种或多种酶的抑制剂在制备预防或治疗急性淋巴细胞白血病耐药复发的药物中的应用及一种评估急性淋巴细胞白血病耐药复发风险的试剂盒及其在评估儿童ALL耐药复发风险中的应用。本发明对后续临床的复发早期的基因诊断和个体化治疗有重要的指导意义。
本发明解决上述技术问题的技术方案如下：
本发明技术方案之一：PRPS1作为急性淋巴细胞白血病耐药复发的基因标志物的用 途。
本发明还提供了一种PRPS1突变体，所述PRPS1突变体的氨基酸序列是序列表中SEQ ID No.2所示的序列中下述位点发生氨基酸的替换突变后形成，所述替换为：第103位丝氨酸替换为苏氨酸、第103位丝氨酸替换为天冬酰胺、第144位天冬酰胺替换为丝氨酸、第176位赖氨酸替换为天冬酰胺、第183位天冬氨酸替换为谷氨酸、第190位丙氨酸替换为苏氨酸、第191位亮氨酸替换为苯丙氨酸、第303位苏氨酸替换为丝氨酸、第53位缬氨酸替换为丙氨酸、第72位异亮氨酸替换为缬氨酸、第77位半胱氨酸替换为丝氨酸、第139位天冬氨酸替换为甘氨酸、第311位酪氨酸替换为半胱氨酸、第103位丝氨酸替换为异亮氨酸、第114位天冬酰胺替换为天冬氨酸或第174位甘氨酸替换为谷氨酸。所述第103位丝氨酸替换为苏氨酸的突变体在本发明中被表示为S103T，其他突变体的表达方式与此相同，依次为S103N、N144S、K176N、L191F、D183E、A190T、T303S、V53A、I72V、C77S、D139G、Y311C、S103I、N114D或G174E。
本发明提供一种PRPS1突变基因，所述突变基因编码上述氨基酸序列的PRPS1突变体。
本发明中，所述的PRPS1突变基因，可以如本领域常规，由于碱基的简并性，只要能够编码上述氨基酸序列的PRPS1突变体即可。所述突变基因的DNA序列较佳地为如序列表SEQ ID No.1所示的序列中下述位点发生核苷酸的替换突变后形成，所述替换为：第308位G替换为C、第308位G替换为A、第431位A替换为G、第528位G替换为C、第549位C替换为G、第568位G替换为A、第573位G替换为C、第908位C替换为G、第158位T替换为C、第214位A替换为G、第230位G替换为C、第416位A替换为G、第932位A替换为G、第308位G替换为T、第340位A替换为G或第521位G替换为A。
所述第308位G替换为C在本发明中表示为G308C，其他替换与此类似，分别为G308A、A431G、G528C、C549G、G568A、G573C、C908G、T158C、A214G、G230C、A416G、A932G、G308T、A340G或G521A。
本发明提供一种包含如本发明所述PRPS1突变基因的重组载体。所述重组载体为本领域常规，较佳地为能表达所述PRPS1突变基因的原核表达载体或真核表达载体，更佳地为能表达所述PRPS1突变基因的慢病毒表达载体，最佳地为GV303慢病毒表达载体(吉凯基因)。所述真核表达载体的制备方法为本领域常规，较佳地为通过下述方法制得：将慢病毒表达载体用限制性内切酶线性化，通过clontech公司的In-FusionTMPCR Cloning Kit试剂盒将将PCR扩增PRPS1突变基因所得的产物交换入所述线性化的慢病毒载体， 通过大肠杆菌TOP10扩增重组子，形成含有本发明PRPS1突变基因的重组载体。
本发明提供一种包含所述重组载体的转化体。所述转化体为本领域常规，只要能满足重组载体可稳定地自行复制，且所携带的本发明的PRPS1突变基因可被有效表达即可，较佳地为表达本发明PRPS1突变基因的原核细胞或真核细胞，更佳地为表达本发明PRPS1突变基因的真核细胞，最佳地为表达本发明PRPS1突变基因的Reh细胞系；所述转化体的制备方法为本领域常规，较佳地为使用表达本发明PRPS1突变基因的慢病毒载体包装成为慢病毒后感染所述真核细胞制得，最佳地为使用表达本发明PRPS1突变基因的慢病毒载体GV303(吉凯基因)与病毒包装的辅助质粒共转HEK293T细胞系包装成慢病毒后感染真核细胞Reh，再经流式细胞仪分选绿色荧光阳性的细胞制得。
本发明技术方案之二：一组评估急性淋巴细胞白血病耐药复发风险的PRPS1突变体群，所述PRPS1突变体群包括以下PRPS1突变体：其氨基酸序列如序列表中SEQ ID No.2所示的序列中第103位丝氨酸替换为苏氨酸、第103位丝氨酸替换为天冬酰胺、第144位天冬酰胺替换为丝氨酸、第176位赖氨酸替换为天冬酰胺、第183位天冬氨酸替换为谷氨酸、第190位丙氨酸替换为苏氨酸、第191位亮氨酸替换为苯丙氨酸或第303位苏氨酸替换为丝氨酸，即S103T、S103N、N144S、K176N、L191F、D183E、A190T或T303S。
所述突变体群较佳地还可以包括以下PRPS1突变体中的一种或多种：其氨基酸序列如序列表中SEQ ID No.2所示的序列中第53位缬氨酸替换为丙氨酸、第72位异亮氨酸替换为缬氨酸、第77位半胱氨酸替换为丝氨酸、第139位天冬氨酸替换为甘氨酸、第311位酪氨酸替换为半胱氨酸、第103位丝氨酸替换为异亮氨酸、第114位天冬酰胺替换为天冬氨酸、第174位甘氨酸替换为谷氨酸或第190位甘氨酸替换为缬氨酸。即V53A、I72V、C77S、D139G、Y311C、S103I、N114D、G174E或A190V。
本发明提供了一组评估急性淋巴细胞白血病耐药复发风险的PRPS1突变基因群，所述PRPS1突变基因群包括以下PRPS1突变基因：其核苷酸序列如序列表中SEQ ID No.1所示的序列中第308位G替换为C、第308位G替换为A、第431位A替换为G、第528位G替换为C、第549位C替换为G、第568位G替换为A、第573位G替换为C或第908位C替换为G。即G308C、G308A、A431G、G528C、C549G、G568A、G573C或C908G。
所述突变基因群较佳地还可以包括以下PRPS1突变基因中的一种或多种：其核苷酸序列如序列表中SEQ ID No.1所示的序列中第158位T替换为C、第214位A替换为G、第230位G替换为C、第416位A替换为G、第932位A替换为G、第308位G替换为T、第340位A替换为G、第521位G替换为A或第569位C替换为T。即T158C、 A214G、G230C、A416G、A932G、G308T、A340G、G521A或C569T。
本发明提供了一种评估ALL耐药复发风险的试剂盒，所述试剂盒包括检测本发明所述突变基因群中的PRPS1突变基因的试剂和使用说明书。
所述试剂可为本领域常规的任何检测PRPS1突变基因的试剂，较佳地包括扩增PRPS1基因各外显子的引物、DNA聚合酶、dNTP或缓冲液中的一种或多种。
所述扩增PRPS1基因各外显子的引物是本领域常规的可扩增PRPS1基因各外显子的引物，较佳地其核苷酸序列如序列表中SEQ ID NO.3～SEQ ID NO.16中任一项所示。
所述检测试剂盒较佳地还可以包括提取细胞或组织样本DNA的试剂、所述提取细胞或组织样本DNA的试剂为本领域常规，较佳地为蛋白酶、饱和酚、体积比为24：1的氯仿和异戊醇混合液、醋酸钠、无水乙醇、70％乙醇和TE溶液，所述百分比为体积百分比，更佳地为Qiagen公司生产的DNA抽提试剂盒。
所述DNA聚合酶为本领域常规，较佳地为TOYOBO公司生产的KOD-Plus DNA聚合酶。
所述dNTP为本领域常规，较佳地为dATP、dGTP、dCTP和dTTP四种的混合物。
所述缓冲液为与所述DNA聚合酶相配套的缓冲体系，较佳地为TOYOBO公司生产的KOD-Plus DNA聚合酶缓冲液。
所述使用说明书记载着试剂盒的使用步骤，较佳的所述使用说明书记载的内容包括以下步骤：：
(1)抽提样本基因组DNA；
(2)检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因。
步骤(2)中，所述检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因的方法为本领域常规，较佳地以步骤(1)所得的样本基因组DNA为模板PCR扩增PRPS1基因各外显子，将扩增片段测序。
所述使用说明书较佳地还可以包括步骤(3)：检测到样本基因组DNA中存在所述PRPS1突变基因群中的任意一种或多种PRPS1突变基因，则该样本有急性淋巴细胞白血病耐药复发的风险。若存在所述的PRPS1突变基因，则认为样本有耐药复发的风险。
本发明提供了一种评估病人ALL耐药复发风险的检测方法，包括下述步骤：
(1)抽提样本基因组DNA；
(2)检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因。
步骤(1)中，所述样本为本领域常规，较佳地为待检病人的骨髓来源或外周血来源的肿瘤细胞或体外培养的永生化细胞系。
步骤(1)中，所述抽提样本基因组DNA为本领域常规，较佳地为使用Qiagen公司生产的DNA抽提试剂盒抽提。
步骤(2)中，所述检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因的方法为本领域常规，较佳地包括：以步骤(1)所得的样本基因组DNA为模板PCR扩增PRPS1基因各外显子，将扩增片段测序。所述PCR扩增为本领域常规，较佳地为使用下述条件进行扩增：
PCR具体体系：
10×KOD缓冲液：  2μl
2mMdNTP:         2μl
25mMMgSO4:       0.8μl
正向引物(10pmol):0.5μl
反向引物(10pmol):0.5μl
基因组DNA模板：  1μl(50ng)
无菌水：         12.7μl
KOD-PLUS聚合酶：0.5μl
PCR具体反应程序：
95℃  10分钟  预变性
95℃  10秒   变性
55℃  30秒   退火
68℃  15秒   延伸
30个循环
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所述测序为本领域常规，较佳地为一代测序或二代测序。
所述检测方法较佳地还可以包括步骤(3)：按以下标准评估急性淋巴细胞白血病耐药复发的风险：检测到样本基因组DNA中存在所述PRPS1突变基因群中的任意一种或多种PRPS1突变基因，则该样本有急性淋巴细胞白血病耐药复发的风险。
本发明技术方案之三：一种评估急性淋巴细胞白血病耐药复发风险的试剂盒，其包括：检测所述PRPS1突变体群中的PRPS1突变体的试剂和使用说明书。
所述试剂为本领域常规，较佳地为检测PRPS1突变体酶活性的试剂，更佳地为包括用同位素碳原子标记的葡萄糖和PRPP。
所述同位素碳原子标记的葡萄糖为本领域常规，较佳地为用同位素13C标记的葡萄 糖，所述PRPP为本领域常规，较佳地为购自Sigma公司的PRPP。
所述试剂盒较佳地还可包括细胞裂解试剂，所述细胞裂解试剂为本领域常规，较佳地为80％甲醇，所述百分比为体积百分比。
所述试剂盒较佳地还可包括无葡萄糖培养基，所述无葡萄糖培养基为本领域常规，较佳地为购自Gibco公司的无葡萄糖培养基。
所述使用说明书记载了试剂盒的使用说明，记载的内容较佳的包括以下步骤：
(1)裂解样本细胞；
(2)检测步骤(1)所得的细胞裂解液中的PRPS1突变体。
其中，步骤(1)所述裂解样本细胞是本领域常规的。步骤(2)所述检测步骤(1)所得的细胞裂解液中的PRPS1突变体的方法是本领域常规，较佳地是检测PRPS1的酶活性。所述使用说明书记载的内容较佳的还包括步骤(3)：检测到样本中存在酶活性高于野生型PRPS1的PRPS1突变体，则该样本有急性淋巴细胞白血病耐药复发的风险。所述PRPS1突变体较佳地为本发明技术方案之二中所述PRPS1突变体群中的任意一种或多种。
本发明提供了一种通过检测PRPS1酶活性来评估儿童ALL耐药复发风险的方法，所述方法包括下述步骤：
(1)裂解样本细胞；
(2)检测步骤(1)所得的细胞裂解液中的PRPS1突变体。
较佳的，所述方法包括下述步骤：
(a)分离ALL病人的白血病细胞；
(b)培养步骤(a)中所述白血病细胞；
(c)用化疗药物处理步骤(b)所述白血病细胞；
(d)收集步骤(c)所述白血病细胞，裂解；
(e)通过LC-MS检测PRPP含量。
步骤(c)中，所述化疗药物为本领域常规，较佳地为嘌呤类似物类化疗药物，更佳地为6-MP或6-TG。
步骤(d)中，所述裂解为本领域常规，较佳地为使用80％甲醇裂解。
步骤(e)中，所述检测为本领域常规，较佳地为使用LC-MS检测PRPP含量。
本发明技术方案之四：一种检测治疗或预防儿童ALL耐药复发的药物活性的方法，其包括以下步骤：
(1)使化合物与含所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，得预处理的白血病细胞；
(2)使抗白血病的化疗药物与步骤(1)所得的预处理的白血病细胞接触。
所述方法较佳地还可以包括步骤(3)，步骤(3)包括：检测步骤(2)中所得的细胞的存活率。
本发明提供了一种筛选治疗或预防儿童急性淋巴细胞白血病耐药复发的药物的方法，其包括下述步骤：
(1)使候选化合物与含所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，得预处理的白血病细胞；
(2)使抗白血病的化疗药物与步骤(1)所得的预处理的白血病细胞接触。
本发明中，步骤(1)所述表达本发明PRPS1突变基因的白血病细胞为本领域常规，较佳地为人源细胞，更佳地为人源淋巴细胞，最佳地为Reh细胞或Jurkat细胞。所述候选化合物为本领域常规，较佳的为现有的化合物、天然产物或核酸类药物，更佳地为靶向嘌呤合成途径的化合物、天然产物或核酸类药物；所述候选化合物为具有潜在的逆转ALL耐药复发的效果的药物。
所述接触为本领域常规，较佳地为使所述候选化合物能够直接接触所述的白血病细胞，更佳地为将所述候选化合物直接加到所述白血病细胞的培养基中。
本发明中，步骤(2)所述处理为本领域常规，较佳地为使所述化疗药物能够直接接触所述的白血病细胞，更佳地为将所述化疗药物直接加到所述白血病细胞的培养基中。所述化疗药物较佳地为嘌呤类药物，所述嘌呤类药物较佳的为6-MP或6-TG。
较佳的，步骤(1)中还同时不采用候选化合物与含如权利要求9或10所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，作为对照。
本发明中，较佳的还包括步骤(3)，即检测步骤(2)所得的细胞的存活率。步骤(3)所述检测的方法为本领域常规，较佳地为使用活细胞染料染色后计数法、MTT法或通过测量荧光素酶与存活细胞内的ATP结合后发出荧光强度的方法，更佳地为使用市售可得的商品化检测试剂盒来检测，如Promega公司的Cell Titer-Glo试剂。
较佳的，步骤(3)同时检测步骤(2)中所述对照中的细胞的存活率。对存活率进行比较，如果测试细胞的存活率明显低于对照细胞，可被认为待检测药物具有逆转ALL耐药复发的活性。
本发明中，所述“耐药复发”是指急性淋巴细胞白血病病人在恢复期对化疗药物的治疗产生耐药性，所述化疗药物为本领域常规，较佳地为嘌呤类化疗药物，更佳地为6-巯基嘌呤(6-MP)或6-巯鸟嘌呤(6-TG)。
所述“预防”是指在可能存在所述耐药复发的情况下，使用后防止或降低所述耐药复 发的发生。
所述“治疗”是指减轻所述耐药复发的程度，或者治愈所述耐药复发使急性淋巴细胞白血病病人恢复正常，或者减缓所述耐药复发的进程。
本发明技术方案之五：一种预防或治疗急性淋巴细胞白血病耐药复发的药物的靶标，所述靶标为嘌呤从头合成途径中所有酶中的一种或多种。
本发明中，所述靶标较佳地为嘌呤从头合成途径中选自PRPS1、PRPS2、PPAT、PFAS、GART、ATIC、PAICS和ADSL中的一种或多种
本发明技术方案之六：一种siRNA，所述的siRNA包括正义RNA片段与反义RNA片段，所述正义RNA片段包含权利要求1所述靶标的DNA编码的RNA分子，所述正义RNA片段与反义RNA片段能够互补形成双链RNA分子。
本发明技术方案之七：一种向导RNA，其针对的靶标为GART基因，其核苷酸序列如SEQ.ID No.41所示。
本发明技术方案之八：一种慢病毒载体，其特征在于，其含有如本发明技术方案之七所述向导RNA的DNA序列和Cas9基因。
本发明中，所述慢病毒载体的空载为本领域常规，较佳地为基于HIV-1的慢病毒载体，更佳地为购自addgene的lentiCRISPR载体。
本发明技术方案之九：一种慢病毒，其由如本发明技术方案之八所述慢病毒载体在慢病毒包装质粒的辅助下在细胞系中经病毒包装而成。
本发明中，所述慢病毒包装质粒为本领域常规，较佳地为psPAX2与pMD2.G，所述细胞系为本领域常规，较佳地为HEK293T细胞系。
本发明中，所述病毒包装为本领域常规，较佳地为将所述下调GART基因表达的慢病毒载体与所述慢病毒包装质粒一起共转到所述细胞系中，培养后收集培养基上清浓缩得到。所述浓缩为本领域常规，较佳地为通过Amicon Ultra-15100KD的超滤管(Millipore)，4℃离心30分钟浓缩收集的病毒液。
本发明技术方案之十：一种向导RNA，其针对的靶标为ATIC基因，其核苷酸序列如SEQ.ID No.42所示。
本发明技术方案之十一：一种慢病毒载体，其特征在于，其含有如本发明技术方案之十所述向导RNA的DNA序列和Cas9基因。
本发明中，所述慢病毒载体的空载为本领域常规，较佳地为基于HIV-1的慢病毒载体，更佳地为购自addgene的lentiCRISPR载体。
本发明技术方案之十二：一种慢病毒，其由如本发明技术方案之十一所述慢病毒载 体在慢病毒包装质粒的辅助下在细胞系中经病毒包装而成。
本发明中，所述慢病毒包装质粒为本领域常规，较佳地为psPAX2与pMD2.G，所述细胞系为本领域常规，较佳地为HEK293T细胞系。
本发明中，所述病毒包装为本领域常规，较佳地为将所述下调ATIC基因表达的慢病毒载体与所述慢病毒包装质粒一起共转到所述细胞系中，培养后收集培养基上清浓缩得到。所述浓缩为本领域常规，较佳地为通过Amicon Ultra-15100KD的超滤管(Millipore)，4℃离心30分钟浓缩收集的病毒液。
本发明技术方案之八与技术方案之十一中，所述慢病毒载体的Cas9基因为本领域常规，较佳地为按照人类遗传密码子优化后的Cas9基因。
本发明技术方案之十三：一种如本发明技术方案之七和/或之十所述向导RNA、如本发明技术方案之八和/或之十一所述慢病毒载体或如本发明技术方案之九和/或之十二所述慢病毒在制备治疗或预防急性白血病耐药复发的药物中的应用。
本发明技术方案之十四：一种治疗或预防急性淋巴细胞白血病耐药复发的药物组合物，其有效物质含有选自如本发明技术方案之六所述siRNA、如本发明技术方案之七和/或之十所述向导RNA、如本发明技术方案之八和/或之十一所述慢病毒载体和如本发明技术方案之九和/或之十二所述慢病毒中的一种或多种。
本发明技术方案之十五：一种嘌呤从头合成途径中所有酶中的一种或多种酶的抑制剂在制备预防或治疗急性淋巴细胞白血病耐药复发的药物中的应用。
本发明中，所述抑制剂为本领域常规，较佳地为抑制性RNA、抑制性多肽、抗体和小分子化合物抑制剂中的一种或多种，更佳地为抑制性RNA和小分子化合物抑制剂中的一种或多种。所述抑制性RNA为本领域常规，较佳地为选自向导RNA、siRNA、shRNA、miRNA、反义RNA和核酶中的一种或多种，更佳地为选自向导RNA、siRNA和shRNA中的一种或多种，最佳地为向导RNA，如序列如序列表SEQ ID NO.41或SEQ ID NO.42所示的向导RNA。所述小分子化合物抑制剂为本领域常规，较佳地为选自洛美曲沙(Lometrexol)和力比泰(Alimta)中的一种或两种，更佳地为洛美曲沙。
本发明中，所述“耐药复发”是指急性淋巴细胞白血病病人在恢复期对化疗药物的治疗产生耐药性，所述化疗药物为本领域常规，较佳地为嘌呤类化疗药物，更佳地为6-巯基嘌呤(6-MP)或6-巯鸟嘌呤(6-TG)。
所述“预防”是指在可能存在所述耐药复发的情况下，使用后防止或降低所述耐药复发的发生。
所述“治疗”是指减轻所述耐药复发的程度，或者治愈所述耐药复发使急性淋巴细 胞白血病病人恢复正常，或者减缓所述耐药复发的进程。
所述预防或治疗急性淋巴细胞白血病耐药复发的药物包括所述抑制性RNA、抑制性多肽、抗体和小分子化合物抑制剂中的一种或多种和一种药用载体。在所述药物中，所述抑制性RNA、抑制性多肽、抗体和小分子化合物抑制剂中的一种或多种可以单独或和其他组分一起作为活性成分。所述活性成分是指具有预防或治疗所述耐药复发的功能。所述的药用载体包括药学上可接受的赋形剂、填充剂、稀释剂等。
所述的药物中，所述抑制性RNA、抑制性多肽、抗体和小分子化合物抑制剂中的一种或多种的含量是0.01～99.99％，所述药用载体的含量是0.01～99.99％，所述百分比是占药物总质量的质量百分比。
所述的药物的剂型没有特别限制，可以是固体、半固体或液体的形式，可以是水溶液、非水溶液或混悬液，也可以是片剂、胶囊、颗粒剂、注射剂或输注剂等。可以经口服途径应用，也可以经静脉、肌肉、皮内或皮下注射途径给药。
本发明中，所述预防或治疗急性淋巴细胞白血病耐药复发的药物在治疗时的使用剂量根据患者的年龄和病情而定，常用的每日剂量约为0.0001～1000mg/kg体重，较佳地为0.01～500mg/kg体重，更佳地为0.1～200mg/kg体重。给药次数一天一次或数次。
本发明中，所述预防或治疗急性淋巴细胞白血病耐药复发的药物在治疗时还可以与化疗药物配合使用。所述化疗药物为本领域常规，较佳地为嘌呤类化疗药物，更佳地为6-巯基嘌呤(6-MP)或6-巯鸟嘌呤(6-TG)。
本发明技术方案之十六：一种评估急性淋巴细胞白血病耐药复发风险的试剂盒，其裂解样本细胞的试剂、检测次黄嘌呤(HX)、次黄嘌呤核苷酸单磷酸(IMP)、5－氨基咪唑－4－甲酰胺核苷酸(AICAR)和次黄苷(Inosine)中一种或多种的试剂和使用说明书；所述使用说明书记载的内容包括以下步骤：
(a)收集、裂解样本细胞；
(b)检测步骤(a)所得的样本细胞裂解液中的选自次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的含量；
(c)通过步骤(b)所得的次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的浓度判断样本急性淋巴细胞白血病耐药复发的风险。
本发明中，所述试剂盒较佳地还包括标准品，所述标准品为本领域常规，较佳地为为次黄嘌呤、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中的一种或多种，更佳地为5－氨基咪唑－4－甲酰胺核苷酸、次黄苷和全部碳原子为13C、全部氮原子为15N的次黄嘌呤 中的一种或多种。
本发明中，所述裂解样本细胞的试剂为本领域常规，较佳地为80％甲醇，所述百分比为体积百分比。
本发明中，所述检测次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中的一种或多种的试剂为本领域常规，较佳地为质谱检测试剂。
本发明还提供了一种评估急性淋巴细胞白血病耐药复发风险的方法，所述方法包括以下步骤：
(a)收集、裂解样本细胞；
(b)检测步骤(a)所得的样本细胞裂解液中的选自次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的含量；
(c)通过步骤(b)所得的次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的浓度判断样本急性淋巴细胞白血病耐药复发的风险。
本方法中，步骤(a)中所述样本细胞可以为本领域常规的急性淋巴细胞白血病样本细胞，较佳地为含有如前所述PRPS1突变体的急性淋巴细胞白血病样本细胞，更佳地为含有包括如前所述PRPS1突变体的Reh细胞，最佳地为前述Reh-PRPS1-S103T或Reh-PRPS1-A190T细胞；所述裂解可以通过本领域常规的方法进行，较佳地使用80％甲醇裂解所述样本细胞，所述百分比为体积百分比；步骤(b)中，所述检测可以为本领域常规的检测方法，较佳地为质谱检测。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明针对现有的以核苷类似物为化疗药物在治疗急性淋巴细胞白血病会出现耐药性复发的问题，提供了急性淋巴细胞白血病耐药复发相关的突变基因，为治疗ALL耐药复发提供了新的靶点，根据所述突变基因得到的评估ALL耐药复发风险的试剂盒可以实现对儿童急性淋巴细胞白血病耐药复发的风险评估。另外，本发明针对现有的以嘌呤类似物为化疗药物在治疗急性淋巴细胞白血病会出现耐药性复发的问题，提供了一种全新的作用于嘌呤合成途径中的酶类的药物在治疗和预防急性淋巴细胞白血病耐药性复发中的应用。本发明还提供了一类全新的检测急性淋巴细胞白血病耐药复发风险的试剂盒，所述试剂盒检测选自嘌呤合成途径的代谢产物次黄嘌呤核苷酸、次黄嘌呤、AICAR和Inosine中的一种或多种，以此作为评估ALL耐药复发风险的指标，为预防和治疗儿童ALL耐药性复发提供了有力的技术手段和支持。
附图说明
图1：超深度测序发现PRPS1为复发特异性突变且突变比例在临床复发前迅速增加。
图2：PRPS1突变蛋白结构示意图。
图3：pET28a载体图谱。
图4：纯化得到的PRPS1野生型及各突变体SDS-PAGE电泳结果。
图5：PCR扩增得到PRPS1野生型琼脂糖凝胶电泳结果。
图6：表达PRPS1野生型及各突变体的稳定Reh细胞系Western结果。
图7：表达PRPS1野生型及各突变体的稳定Reh细胞系对6-MP和6-TG的药物敏感性结果。
图8：表达PRPS1野生型及各突变体的稳定Reh细胞系对6-MP和6-TG的细胞凋亡结果。
图9：细胞内化疗药物6-MP/6-TG的代谢产物检测结果。
图10：表达PRPS1野生型及各突变体的PRPS1酶活性检测结果。
图11：ADP和GDP对PRPS1反馈调节路径示意。
图12：GDP/ADP对PRPS1蛋白活性的抑制的效果。
图13：细胞内嘌呤代谢途径产物含量测定结果。
图14：靶向嘌呤合成途径的核酸药物CRISPR向导RNA对PRPS1S103T与A190T突变体耐药性的抑制作用。
图15：外源加入嘌呤影响Reh细胞对化疗药物6-MP的药物耐受。
图16：外源加入嘌呤影响Reh细胞对化疗药物6-MP的代谢。
图17：次黄嘌呤竞争性抑制化疗药物6-MP的反应。
图18：洛美曲沙(Lometrexol)逆转PRPS1基因突变导致的6-MP药物耐受。
图19：靶向嘌呤合成途径的核酸药物shRNA对PRPS1S103T与A190T突变体耐药性的抑制作用。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明通过全外显子组测序技术，对16组儿童ALL的初发、缓解和复发样本测序， 发现嘌呤合成限速酶PRPS1(Phosphoribosyl pyrophosphate synthase I)基因存在多个位点的复发特异性突变，在初发与缓解样本中均为野生型。通过进一步对144例复发样本进行了PRPS1测序，又发现16例复发样本中出现了PRPS1基因的突变，在B-ALL中突变频率为13％(18/138)。同时德国的夏里特医学院(Charité-
Figure PCTCN2016073906-appb-000002
Berlin)儿童血液肿瘤中心的Renate Kirschner-Schwarb博士的研究也证实了在德国儿童ALL中PRPS1存在复发特异性突变，突变比例为2.7％(6/220)。针对所有突变中最主要的四类突变A190T、T303S、K176N和N144S，通过对一系列骨髓样本的超深度测序发现，所有的样本中的病人在诊断期在超深度测序的分辨率范围内均未出现上述突变。此外，这些PRPS1的突变在临床复发期之前被发现呈指数增长，如图1所示。在PRPS1野生型的ALL细胞系Reh中过表达所述PRPS1的突变体，通过药物敏感性实验发现，复发性特异的PRPS1基因突变使Reh对6-MP/6-TG产生了耐药，证明PRPS1基因突变在儿童ALL耐药复发过程中具有重要的作用。
本发明结合细胞生物学、分子生物学、代谢组学等方法，从PRPS1基因突变对自身酶活性的影响，以及对6-MP药物代谢和嘌呤代谢网络的影响等方面系统地研究PRPS1基因突变介导儿童ALL耐药复发的机制。研究揭示了儿童ALL一种新的耐药复发机制，提示PRPS1突变可以驱动复发，是一个耐药复发的基因标志物，并对后续临床的复发早期的基因诊断和个体化治疗有重要的指导意义。
以下实施例中所涉及的D183H突变体为已报道过的PRPS1基因的功能获得性突变体；所涉及的A87T与M115T突变体为已报道的功能缺失性突变体，它们分别作为检验实验检测的体系是否正常的正对照与负对照。所涉及的S103T、S103N、N144S、T303S、K176N、D183E、A190T、A190V和L191F为实验组，这些突变体通过比对人PRPS1的晶体结构可以被分为两类，即位于PRPS1二聚体形成界面的突变位点与位于PRPS1变构位点的突变位点，如图2所示。组内的突变体为PRPS1基因的耐药突变体。
实施例1
检测样本中的PRPS1基因突变:
通过全外显子组测序技术，对16组儿童ALL的初发、缓解和复发样本及144例复发样本进行了PRPS1的测序。
样品的质控：样品质量直接关系到测序结果的可靠性，通过以下三种方法保证用于深度测序和后续验证样本的质量：①白血病初发和复发时其骨髓中的白血病细胞可占全部有核细胞的90％以上，通过Ficoll密度梯度离心可以去除残余的正常粒细胞和有核红细胞，从而可以得到高纯度的白血病细胞用于后续的DNA抽提和测序；②通过多色荧 光抗体组合可以区分白血病细胞和正常骨髓造血细胞，对于个别纯度较低的样品可通过流式细胞技术分选得到高纯度(>99％)的样品；③根据微量残留病检测结果，选择残留白血病细胞低于0.01％的缓解期样本用于深度测序，从而可以保证初发和复发特异性突变的可靠性。
样品的制备：通过QIAGEN Blood DNA kit进行白血病细胞基因组DNA的抽提，Q-bit荧光定量试剂盒进行DNA浓度测定，将高纯度基因组DNA用于后期的深度测序。
PRPS1各外显子的扩增：针对PRPS1基因组序列设计合适的引物进行PRPS1各外显子的扩增，所述PCR体系与程序如下所示：
PCR具体体系：
10×KOD缓冲液：  2μl
2mM DNTP:        2μl
25mM MgSO4:      0.8μl
正向引物(10pmol):0.5μl
反向引物(10pmol):0.5μl
基因组DNA模板：  1μl(50ng)
无菌水：12.7μl
KOD-PLUS聚合酶：0.5μl
PCR具体反应程序：
95℃  10分钟  预变性
95℃  10秒  变性
55℃  30秒  退火
68℃  15秒  延伸
68℃  10分钟
引物序列如表1所示，PCR产物按常规进行毛细管电泳测序和突变分析，突变分析使用软件为Mutation Surveyor。
测序分析：PCR成功后将目的条带测序，将目的序列与NCBI中的PRPS1基因序列进行比对，分析PRPS1的基因突变情况。实验结果见表2与表3。
通过对16组儿童ALL的初发、缓解和复发样本测序，发现嘌呤合成限速酶PRPS1(Phosphoribosyl pyrophosphate synthase I)基因存在多个位点的复发特异性突变(见表2)，在初发与缓解样本中均为野生型。通过对144例复发样本进行了PRPS1测序，又发现16例复发样本中出现了PRPS1基因的突变，突变频率为13％(18/138)。同时德国的夏里特 医学院(Charité-
Figure PCTCN2016073906-appb-000003
Berlin)儿童血液肿瘤中心的Renate Kirschner-Schwarb博士的研究也证实了本发明的结果，在德国儿童ALL中PRPS1存在复发特异性突变，突变比例为2.7％(6/220)(见表2)。结合临床病理学分析显示，存在PRPS1基因突变的病人均为早期复发(P<0.005)(见表3)。超深度测序发现PRPS1为复发特异性突变且突变比例在临床复发前迅速增加。意味着PRPS1驱动了病人的耐药复发，可以作为临床复发诊断的基因标志物以及临床复发治疗的一个重要靶点。
表1PCR扩增外显子信息
Figure PCTCN2016073906-appb-000004
表2中国和德国儿童B-ALL复发样本中的PRPS1突变
Figure PCTCN2016073906-appb-000005
Figure PCTCN2016073906-appb-000006
通过全外显子组测序技术对儿童ALL初发、缓解、复发骨髓样本进行测序，发现了18例病人复发样本中存在PRPS1基因突变，突变频率为13％(18/138)，如表2所示。其中有9例复发样本中均为A190T的位点突变。通过与德国的夏里特医学院(Charité-
Figure PCTCN2016073906-appb-000007
Berlin)儿童血液肿瘤中心的Renate Kirschner-Schwarb博士合作也证 实了在德国儿童ALL中PRPS1存在复发特异性突变，突变比例为2.7％(6/220)。
表3中国和德国B-ALL患儿中PRPS1突变与临床特征的关系
Figure PCTCN2016073906-appb-000008
Figure PCTCN2016073906-appb-000009
Figure PCTCN2016073906-appb-000010
1P值用Fisher精确检验计算得到。
2P值用卡平方检验计算得到。
Figure PCTCN2016073906-appb-000011
复发时间:较早期,诊断后的18个月之内；早期,诊断后的18个月与36个月之间；后期,初次治疗后的36个月之后。
如表3所示，通过临床病理学资料，分析发现PRPS1突变都发生在早期复发病人中(中国病人，P<0.005，德国病人，P<0.001)，预示具有缓解期诊断复发意义。
实施例2
检测样本中的嘌呤代谢途径相关基因的突变
通过常规的二代测序技术，在160例儿童ALL复发样本中进行了嘌呤代谢相关酶HGPRT、IMPDH、NT5C2、PRPS2、ATIC、ADSL、GART、PFAS的测序。
样品的质控、制备、基因外显子的扩增同实施例1
测序分析：PCR成功后将目的条带测序，将目的序列与NCBI中对应的基因序列进行比对，分析基因突变情况。如果复发样本中发现了基因突变，检测同一病人的初发样本，确定是否是复发特异性突变。实验结果见表4。
通过测序与序列比对发现，嘌呤代谢相关酶PRPS2、ATIC、ADSL、GART、PFAS均存在复发特异性突变。
表4嘌呤代谢相关酶在儿童ALL复发样本中的突变
Figure PCTCN2016073906-appb-000012
Figure PCTCN2016073906-appb-000013
实施例3
PRPS1原核表达载体的构建
1.目的基因片段的获取：根据NCBI提供的PRPS1(gene ID:5631,NM 002764)序列，设计野生型PRPS1的PCR引物序列，正向引物：5’ cgcggcagccatATGCCGAATATCAAAATCTTCAG 3’，反向引物：5’gtggtggtgctcgagTTATAAAGGGACATGGCTGAATAGGTA3’(引物说明：含交换配对碱基、酶切位点，并含有目的基因5’端部分序列用于PCR钓取目的基因)。以提取的Reh细胞的cDNA为模板，PCR钓取PRPS1，PCR产物大小为957bp，产物序列如序列表SEQ ID No.1所示。
2.原核表达载体的线性化：用限制性内切酶NdeI/XhoI处理pET28a载体，pET28a载体图谱如图3所示。
3.重组质粒构建：通过clontech公司的In-FusionTMPCR Cloning Kit试剂盒将第1步的PCR产物交换入线性化病毒载体中，通过大肠杆菌TOP10扩增重组子。
4.鉴定重组质粒：通过测序验证重组子是否构建成功。
5.PRPS1突变原核载体的构建：以pET28a-PRPS1为质粒模板，通过TOYOBO公司的KOD-Plus DNA聚合酶进行环状PCR分别构建PRPS1的9个突变体：S103T、S103N、N144S、T303S、K176N、D183E、A190T、A190V和L191F，PCR引物见表5。
PCR具体体系：
10×KOD缓冲液：  2μl
2mM dNTP:        2μl
25mM MgSO4:    0.8μl
正向引物(10pmol):0.5μl
反向引物(10pmol):0.5μl
GV303-PRPS1质粒模板：1μl(10ng)
无菌水：12.7μl
KOD-PLUS聚合酶：0.5μl
PCR具体反应程序：
95℃  10分钟  预变性
95℃  10秒  变性
55℃  30秒  退火
68℃  3分钟  延伸(30秒/Kb)
30个循环
68℃  10分钟
PCR产物加入1μl DpnI酶37℃水浴1小时消化质粒模板后，取10μl加入大肠杆菌TOP10中扩增。转化子经测序验证突变体是否构建成功。
实施例4
PRPS1原核蛋白的纯化
1.pET28a-PRPS1系列质粒转化大肠杆菌BL21(DE3)
①取1μl pET28a-PRPS1系列质粒与100μl BL21(DE3)大肠杆菌感受态细胞混匀，冰上放置30分钟，水浴42℃，90秒，勿摇动，立即置冰上冷却2分钟，加入900μl SOC培养基，37℃，170转/分，振荡1小时.
②将1ml已转化的感受态细胞，涂在含50μg/ml卡那霉素的半固体LB琼脂培养皿中倒置平皿培养16小时，出现菌落，拟挑出长势良好的阳性克隆做进一步实验。
2.小量表达测试：
①从上述pET28a-PRPS1系列转化平板中，挑取单克隆到3ml LB(含抗生素)中，37℃，220rpm振荡培养10～12小时。
②次日以1:100接菌，即取50μl菌液到5ml LB(含抗生素)中，37℃，220rpm振荡培养2～3小时至OD值达到0.6。
③对照取样：取2ml菌液，12000rpm离心1分钟，弃尽上清，菌体沉淀中加入1ml PBS溶液，超声破碎(200w，超声3秒,间歇5秒,20循环)，加入SDS-PAGE Loading Buffer(还原,4x),100℃,水浴10分钟，12000rpm离心10分钟，取上清，-20℃保存，做为诱导前对照。
④剩余3ml菌液中，加诱导剂IPTG至终浓度为1mM，16℃，220rpm振荡培养16小时后，12000rpm离心1分钟，弃尽上清收菌，超声破碎处理取样，-20℃保存。
⑤对上述样品进行SDS-PAGE电泳鉴定表达结果。
3.PRPS1大量表达：
①挑单克隆到100ml LB(含50μg/ml卡那霉素抗生素)中，37℃，220rpm振荡培养10～12小时。
②扩大培养：以1:100接菌，即各取上述20ml菌液接到2L LB培养基中(含终浓度50μg/ml卡那霉素抗生素)中，37℃，220rpm振荡培养4～5小时至OD值达到0.6-0.8。
③对照取样：取2ml菌液，12000rpm离心1分钟，弃尽上清，菌体沉淀中加入1ml PBS溶液，超声破碎(200w，超声3秒,间歇5秒,20循环)，加入SDS-PAGE Loading Buffer(还原,4x),100℃,水浴10分钟，12000rpm离心10分钟，取上清，-20℃保存，做为诱导前对照。
④其余菌液于16℃，220rpm继续振荡培养1小时后，加IPTG至终浓度1mM，16℃，220rpm振荡培养12小时后收菌：6000rpm、4℃离心10分钟，弃尽上清，取5ml做表达 鉴定(16℃样品)，其余-80℃保存。
⑤超声破碎分别处理上述PRPS1系列样品，进行SDS-PAGE电泳鉴定。(同小量表达中的5)
4.PRPS1蛋白纯化：分别取超声破碎好的菌液，通过AKTA-purifier系统的镍柱纯化PRPS1的系列蛋白，通过SDS-PAGE电泳鉴定PRPS1系列蛋白的表达以及纯度，通过碧云天公司的BCA蛋白定量试剂盒对PRPS1系列蛋白进行定量，得到氨基酸序列如序列表中SEQ ID No.2所示的野生型PRPS1蛋白，如图4所示。
实施例5
PRPS1突变基因真核表达载体的制备
1.目的基因片段的获取：根据NCBI提供的PRPS1(gene ID:5631,NM 002764)序列，设计野生型PRPS1的PCR引物序列，正向引物：5’GAGGATCCCCGGGTACCGGTCGCCACCATGCCGAATATCAAAATC 3’，反向引物：5’TCCTTGTAGTCCATACCGTGGTGGTGGTGGTGGTGCTCGAGTAAAG3’，引物含交换配对碱基、酶切位点，并含有目的基因5’端部分序列用于PCR钓取目的基因。以pET-28a-PRPS1为质粒模板，PCR钓取PRPS1，PCR产物大小为1022bp，如图5所示。
PCR体系：
10×KOD缓冲液     ：  2μl
2mM dNTP          :  2μl
25mM MgSO4        :  0.8μl
正向引物(10pmol)  :  0.5μl
反向引物(10pmol)  :  0.5μl
pET-28a-PRPS质粒  ：  1μl(10ng)
无菌水            ：  12.7μl
KOD-PLUS聚合酶：0.5μl
PCR反应程序：
95℃  10分钟  预变性
95℃  10秒  变性
55℃  30秒  退火
68℃  6分钟  延伸(30秒/Kb)
30个循环
68℃  10分钟
PCR产物加入1μl DpnI酶37℃水浴1小时消化质粒模板后，取10μl加入大肠杆菌TOP10中扩增。转化子经测序验证突变体是否构建成功。
2.病毒表达载体的线性化：用限制性内切酶AgeI处理GV303病毒载体(吉凯基因)。
3.重组质粒构建：通过clontech公司的In-FusionTMPCR Cloning Kit试剂盒按照说明书将第1步的PCR产物交换入线性化病毒载体中，通过大肠杆菌TOP10扩增重组子GV303-PRPS1。
4.酶切鉴定重组质粒：经过限制性内切酶Hind III鉴定重组子是否正确，若产生354bp的酶切片段则为阳性重组子。
5.PRPS1突变质粒的构建：以所述GV303-PRPS1为质粒模板，通过TOYOBO公司的KOD-Plus DNA聚合酶进行环状PCR分别构建PRPS1的9个突变体慢病毒表达载体：GV303-PRPS1-S103T、GV303-PRPS1-S103N、GV303-PRPS1-N144S、GV303-PRPS1-T303S、GV303-PRPS1-K176N、GV303-PRPS1-D183E、GV303-PRPS1-A190T、GV303-PRPS1-A190V和GV303-PRPS1-L191F，PCR引物见表5。
表5PRPS1突变体PCR引物序列表
Figure PCTCN2016073906-appb-000014
Figure PCTCN2016073906-appb-000015
实施例6
表达PRPS1突变基因的Reh细胞的制备
病毒制备
1.转染前24小时将HEK293T细胞种到10cm皿中，第二天当细胞密度达到50％～80％时，开始转染。
2.配制DNA-Opti-MEM和Fugene-6-Opti-MEM混合液(一皿细胞)：
表6
Figure PCTCN2016073906-appb-000016
Promega公司的转染试剂Fugene-6和Opti-MEM混合后，静置5分钟；DNA-Opti-MEM和Fugene-6-Opti-MEM混合后，静置15分钟；
3.混合液静置过程中，从培养箱中取出需转染的HEK293T细胞培养皿，换新鲜的无抗生素培养基。
4.混合液静置15min后，加233μl/皿混合液至HEK293T细胞的培养皿中，“十”字形上下左右混匀培养液5次。
5.37℃，5％CO2条件下培养，24小时后换液，15ml/皿加新鲜培养液，继续培养72小时后，收集含病毒的培养液。
6.将收集的病毒上清液加入Amicon Ultra-15100KD的超滤管，4℃离心30分钟，得到浓缩后的病毒液。
7.将得到的病毒液进行梯度稀释后感染HEK293T细胞，并根据感染后绿色荧光的 细胞数量计算病毒的滴度。
病毒感染
1.细胞接种：将Reh细胞计数后接种到12孔板中，每孔接种3×105个细胞。
2.每孔加入浓缩后的病毒上清(MOI＝10)，同时加入终浓度为8μg/ml的聚凝胺(polybrene)，将细胞放入培养箱继续培养24小时后，换成新鲜的完全培养基。48小时后即可在显微镜下观察荧光。
3.流式分选细胞：病毒感染72小时后，将细胞通过Beckman公司的流式细胞仪Moflo XDP分选出有绿色荧光的Reh细胞，扩大培养。
4.收集分选后的Reh细胞，通过Western blot检测PRPS1野生型与突变型的表达，验证稳定细胞系是否构建成功。实验结果见图6，共获得以下九个稳定细胞系：Reh-PRPS1-S103T、Reh-PRPS1-S103N、Reh-PRPS1-N144S、Reh-PRPS1-T303S、Reh-PRPS1-K176N、Reh-PRPS1-D183E、Reh-PRPS1-A190T、Reh-PRPS1-A190V和Reh-PRPS1-L191F。
用与上述操作相同的方法利用GV303-PRPS1慢病毒表达载体构建得到稳定过表达野生型PRPS1的稳定细胞系Reh-PRPS1-WT。
实施例7
6-MP药物敏感性实验
Reh细胞是一种人急性B淋巴细胞白血病株。通过检测经化疗药物6-MP处理后表达不同PRPS1突变基因的Reh细胞的存活率来检测不PRPS1突变体对化疗药物6-MP的敏感性，步骤如下：
①细胞接种：将表达野生型与各类突变体PRPS1的Reh细胞计数后接种到96孔板中，每孔接种104个细胞，设置5个复孔；
②细胞处理：将药物6-MP进行梯度稀释，起始浓度为100μg/ml，依次3倍稀释，稀释10个梯度，加入铺好细胞的96孔板中，37℃培养72小时；
③细胞活性测定：72小时后，每孔中加入50μl CellTiter-Glo试剂(Promega公司)，室温混匀孵育10分钟，放入酶标仪(Biotek公司)读化学发光值；
④计算IC50：用Graphpad 5.0软件计算药物IC50值，比较组间差异。
实验结果如图7所示。与表达空载的对照相比，实验组各PRPS1突变基因的细胞IC50值都有显著上升，证明表达实验组各PRPS1突变基因的细胞在6-MP处理后存活率显著提高，即耐药性显著增加。
实施例8
6-TG药物敏感性实验
通过检测经化疗药物6-TG处理后表达不同PRPS1突变基因的Reh细胞的存活率来检测不同PRPS1突变体对化疗药物6-TG的敏感性，步骤如下：
①细胞接种：将表达野生型与各类突变体PRPS1的Reh细胞计数后接种到96孔板中，每孔接种104个细胞，设置5个复孔；
②细胞处理：将药物6-TG进行梯度稀释，起始浓度为100μg/ml，依次3倍稀释，稀释10个梯度，加入铺好细胞的96孔板中，37℃培养72小时；
③细胞活性测定：72小时后，每孔中加入50μl CellTiter-Glo试剂(Promega公司)，室温混匀孵育10分钟，放入酶标仪(Biotek公司)读化学发光值；
④计算IC50：用Graphpad 5.0软件计算药物IC50值，比较组间差异。
实验结果如图7所示。与表达空载的对照相比，实验组各PRPS1突变基因的细胞IC50值都有显著上升，证明表达实验组各PRPS1突变基因的细胞在6-TG处理后存活率显著提高，即耐药性显著增加。
实施例9
细胞凋亡的检测实验
①细胞接种：将表达野生型与各类突变体PRPS1的Reh细胞计数后接种到12孔板中，每孔接种3×105个细胞，设置2个复孔；
②细胞处理：将药物6-MP或6-TG 10μg/ml，加入铺好细胞的12孔板中，37℃培养72小时；
③细胞染色：72小时后，离心收集细胞，用PBS缓冲液冲洗细胞，加入BD公司PE标记的Annexin-V与7-AAD染料，室温混匀孵育15分钟，离心弃上清，用PBS冲洗2遍；
④流式检测：用BD公司的流式细胞仪Canto II上机检测细胞凋亡比例。
实验结果如图8所示。与表达空载的对照相比，实验组各PRPS1突变基因的细胞凋亡比例都有显著下调，证明表达实验组各PRPS1突变基因的细胞在6-MP或6-TG处理后存活率显著提高，即耐药性显著增加。
实施例10
检测细胞内化疗药物6-MP/6-TG的代谢产物。
6-MP为前药，在发挥作用之前需要先经过体内代谢反应形成TIMP与TGMP，如图9C所示。检测步骤如下所述：
①细胞接种：将表达PRPS1野生型与各类突变体的Reh细胞计数铺6cm培养皿，每 个皿3×106细胞；
②细胞处理：每个6cm培养皿中加入10μM化疗药物6-MP处理4个小时；
③收集细胞：将细胞转移到离心管中，3,000g离心5分钟，弃上清，按照3×106细胞/200μl 80％甲醇裂解细胞；
④LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测化疗药物6-MP的代谢产物TIMP,TGMP,r-MP,r-TG,r-MMP的含量，分别用TIMP(Jean Bioscience,cat#NU-1148),TGMP(Jean Bioscience,cat#NU-1121),r-MP(Sigma,cat#852686),r-TG(Sigma,cat#858412),r-MMP(Sigma,cat#M4002)做标准曲线定量。
实验结果图9所示，对于实验组经过6-MP处理后的各突变体，6-MP的代谢产物TIMP、TGMP和r-MP、r-TG、r-MMP在胞内的含量与对照相比都显著减少。
实施例11
PRPS1酶活性的检测
本发明结合了细胞生物学与代谢组学的技术，通过同位素标记的方法，检测了PRPS1在细胞中的活性。具体实施方式如下：
①细胞培养：将表达PRPS1野生型与各类突变体的Reh细胞计数铺6cm培养皿，每个皿3×106细胞；
②细胞处理：第二天用无葡萄糖培养基(Gibco,cat#11879-020)悬浮细胞，每个6cm培养皿中加入10mM 13C6-葡萄糖(Cambridge isotope laboratories,cat#CLM-1396-1)标记5分钟；
③收集细胞：将细胞转移到离心管中，3,000g离心5分钟，弃上清，按照3×106细胞/200μl 80％甲醇，裂解细胞；
④LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测13C5-PRPP的含量，用PRPP(sigma,cat#P8296)做标准曲线定量。
实验结果见图10，与表达空载的对照相比，实验组各PRPS1突变基因的催化反应产物PRPP的浓度值都有显著上升，证明表达实验组各PRPS1突变基因的细胞中PRPS1突变体酶的活性都有显著地增加。
实施例12
测定核苷酸ADP、GDP对PRPS1活性的反馈调节
1.体外蛋白水平
①建立PRPS1催化的酶学反应，如图11所示，ATP(货号A7699)、ADP(货号A2754)、GDP(货号G7172)、核糖-5-磷酸(货号R7750)购自sigma公司。反应体系为：50mM Tris  PH7.5，2mM磷酸根，1mM DTT，10mM MgCl2，0.5mM核糖-5-磷酸，0.5mM ATP，PRPS1蛋白。
②测定GDP/ADP对PRPS1蛋白活性的抑制：GDP/ADP起始浓度为5mM，依次做3倍浓度梯度稀释，配制15μL PRPS1体外酶学反应体系加入384孔板，37℃反应30分钟。加入10μl Promega公司的Kinase-Glo试剂(货号V3722)终止反应，室温反应15分钟，在酶标仪中读取化学发光数值。用Graphpad 5.0计算GDP/ADP对PRPS1的活性抑制。
实验结果见图12A、B，可见GDP/ADP对PRPS1突变体S103T、S103N、N144S、T303S、K176N、D183E、A190T、A190V、L191F的抑制明显低于PRPS1野生型，而已知的功能缺失性突变A87T与M115T与野生型无明显差异，即PRPS1的复发特异性突变逃逸了核苷酸GDP/ADP对PRPS1活性的负反馈抑制，为功能获得性突变。
2.细胞水平
本发明结合了细胞生物学与代谢组学的技术，通过同位素标记的方法，检测PRPS1在细胞中的活性，在此基础上评估核苷酸GDP/ADP对PRPS1活性的影响。具体实施方式如下：
①细胞培养：将表达PRPS1野生型与各类突变体的Reh细胞计数铺6cm培养皿，每个皿3×106细胞；
②细胞处理：在培养皿中加入2mM ADP或0.5mM GDP处理4小时后用无葡萄糖培养基(Gibco,cat#11879-020)悬浮细胞，每个6cm培养皿中加入10mM 13C6-葡萄糖(Cambridge isotope laboratories,cat#CLM-1396-1)标记5分钟；
③收集细胞：将细胞转移到离心管中，3,000g离心5分钟，弃上清，按照3×106细胞/200μl 80％甲醇，裂解细胞；
④LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测13C5-PRPP的含量，用PRPP(sigma,cat#P8296)做标准曲线定量。根据PRPP的含量来判定PRPS1酶活性的变化。
实验结果见图12C，表达野生型实验组在加入核苷酸GDP/ADP后PRPP的浓度有所下降，实验组各PRPS1突变基因的催化反应产物PRPP的浓度均不受核苷酸GDP/ADP的影响，证明表达实验组各PRPS1突变基因的细胞中PRPS1突变体酶的活性不受核苷酸GDP/ADP的反馈调节。
实施例13
嘌呤代谢途径活性的检测
本发明结合了细胞生物学与代谢组学的技术，通过同位素标记的方法，检测细胞中嘌呤从头合成途径与补救合成途径的活性。具体实施方式如下：
①细胞培养：将表达PRPS1野生型与各类突变体的Reh细胞计数铺6cm培养皿，每个皿3×106细胞；
②细胞处理：第二天用无氨基酸培养基(Gibco定制,cat#ME100031L1)悬浮细胞，分别用20μg/ml 13C2，15N-甘氨酸(sigma,cat#489522)标记从头合成途径，2μM 13C5，15N4-次黄嘌呤(Cambridge isotope laboratories,cat#489522)标记补救合成途径，分别标记4小时与1小时；
③收集细胞：将细胞转移到离心管中，3,000g离心5分钟，弃上清，按照3×106细胞/200μl 80％甲醇，裂解细胞；
④LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测13C2， 15N–次黄嘌呤核苷酸(IMP+3)与13C5，15N4-次黄嘌呤核苷酸(IMP+9)的含量，用IMP(sigma,cat#I4625)做标准曲线定量。
实验结果如图13、图9B和图18所示。实验组中表达各PRPS1基因耐药突变体的Reh细胞内的13C2， 15N–次黄嘌呤核苷酸(IMP+3)和13C5，15N4-次黄嘌呤核苷酸(IMP+9)的浓度和与表达空载与野生型PRPS1基因的Reh细胞相比有显著的上升，即表明嘌呤从头合成与补救合成两条途径活性均有显著提高。
实施例14
检测样本中的次黄嘌呤、AICAR以及Inosine的含量
本发明结合了细胞生物学与代谢组学的技术，通过LC-MS检测细胞中次黄嘌呤的含量。具体实施方式如下：
①细胞培养：将PRPS1的野生型和各类突变体病毒转染人Reh细胞(此处请参见实施例4)，培养细胞；
②细胞计数：当每个反应孔细胞达到5×106/5ml时，根据实验所需细胞数量进行取样；
③收集细胞：细胞从孔中吸出，1500rpm离心后弃去上清。PBS洗涤1次，再次离心(500g)，吸去上清；
④细胞裂解：按照3×106细胞/200μl 80％甲醇，裂解细胞；
⑤4℃ 14,000g离心10分钟，离心后将上清转移到1.5ml EP管中；
⑥LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测次黄嘌呤、AICAR以及Inosine的含量，分别用13C5，15N4-次黄嘌呤(Cambridge isotope laboratories,cat#489522)、AICAR(sigma,cat#A9978)以及Inosine(sigma,cat#I4125)做 标准曲线绝对定量。
实验结果如图13所示。实验组中表达各PRPS1基因耐药突变体的Reh细胞内的次黄嘌呤、AICAR以及Inosine的浓度与表达空载和野生型PRPS1基因的对照细胞相比都有显著地提高。
实施例15
靶向嘌呤合成途径的核酸药物
针对嘌呤从头合成途径的慢病毒LV-CRISPR-ATIC与LV-CRISPR-GART的制备及其逆转PRPS1基因突变引起的6-MP药物耐受中的应用
①CRISPR慢病毒载体的构建：根据CRISPR设计原则与GART和ATIC的序列，分别设计对应序列如下：CRISPR-ATIC:TGAATCTGGTCGCTTCCGGA(SEQ ID NO.40)，CRISPR-GART:GCAGCCCGAGTACTTATAAT(SEQ ID NO.39)，构建到CRISPR慢病毒载体(Addgene,cat#49535)中，得到慢病毒载体lentiCRISPR-ATIC与lentiCRISPR-GART；
②按已报道的方法(Shalem O,Sanjana NE,Hartenian E,Shi X,Scott DA,Mikkelsen TS,Heckl D,Ebert BL,Root DE,Doench JG,Zhang FScience.2014Jan 3；343(6166):84-7.doi:10.1126/science.1247005.Epub 2013Dec 12)包装获得表达针对ATIC与GART基因的CRISPR RNA的慢病毒LV-CRISPR-ATIC与LV-CRISPR-GART；
③将包装得到的空载慢病毒、慢病毒LV-CRISPR-ATIC与LV-CRISPR-GART分别感染细胞系Reh-PRPS1-S103T与Reh-PRPS1-A190T后形成稳定细胞系，感染Reh细胞的病毒MOI＝10，加入8μg/ml聚凝胺，感染24个小时后换液，培养48小时后加入0.8μg/ml嘌呤霉素抗性筛选一周后形成下述稳定细胞系：对照细胞系Reh-LV-CRISPR、Reh-S103T-CRISPR-ATIC、Reh-S103T-CRISPR-GART、Reh-A190T-CRISPR-ATIC和Reh-A190T-CRISPR-GART。
④细胞处理：将药物6-MP进行稀释，加入铺好如步骤③所述的几种细胞系的96孔板中，每种细胞系设置5个复孔，检测慢病毒感染细胞对6-MP的敏感性变化。具体实施方式同实施例8；
实验结果如图14所示，与空载病毒的对照细胞系Reh-LV-CRISPR相比Reh-S103T-CRISPR-ATIC、Reh-S103T-CRISPR-GART、Reh-A190T-CRISPR-ATIC和Reh-A190T-CRISPR-GART的IC50值都显著下降，即表明对药物6-MP的耐药性显著降低。
实施例16
外源加入嘌呤影响Reh细胞对化疗药物6-MP的药物耐受
通过检测外源加入嘌呤对Reh细胞的药物敏感性，步骤如下：
①细胞接种：将表Reh细胞计数后接种到96孔板中，每孔接种104个细胞，每组设置5个复孔；
②细胞处理：每组分别加入10μM、50μM、100μM的次黄嘌呤(HX)或次黄嘌呤核苷酸(IMP)预处理1小时，然后将药物6-MP进行梯度稀释，起始浓度为100μg/ml，依次3倍稀释，稀释10个梯度，加入铺好细胞的96孔板中，37℃培养72小时；
③细胞活性测定：72小时后，每孔中加入50μl CellTiter-Glo试剂(Promega公司)，室温混匀孵育10分钟，放入酶标仪(Biotek公司)读化学发光值；
④计算IC50：用Graphpad 5.0软件计算药物IC50值，比较组间差异。
实验结果如图15所示。与嘌呤溶剂水对照相比，实验组分别加入次黄嘌呤与雌黄嘌呤核苷酸的细胞IC50值都有显著上升，证明外源加入嘌呤的细胞在6-MP处理后存活率显著提高，即耐药性显著增加。
实施例17
外源加入嘌呤影响Reh细胞对化疗药物6-MP的代谢
①细胞接种：将Reh细胞计数铺6cm培养皿，每个皿3×106细胞；
②细胞处理：每个6cm培养皿中加入50μM的次黄嘌呤(HX)或次黄嘌呤核苷酸(IMP)预处理1小时，后加入10μM化疗药物6-MP处理4个小时；
③收集细胞：将细胞转移到离心管中，3,000g离心5分钟，弃上清，按照3×106细胞/200μl 80％甲醇裂解细胞；
④LC-MS(ABI 5500Qtrap coupled with Waters Acquity UPLC)检测化疗药物6-MP的代谢产物TIMP,TGMP的含量，分别用TIMP(Jean Bioscience,cat#NU-1148),TGMP(Jean Bioscience,cat#NU-1121)做标准曲线定量。
实验结果如图16所示，对于实验组加入外源嘌呤处理后的Reh细胞，6-MP的代谢产物TIMP和TGMP在胞内的含量与对照相比都显著减少。
实施例18
次黄嘌呤竞争性抑制化疗药物6-MP的反应
化疗药物6-MP是次黄嘌呤的类似物，6-MP与次黄嘌呤都是HGPRT的底物，本发明建立了体外酶学反应测定次黄嘌呤与6-MP与HGPRT反应的Km值，反映次黄嘌呤与6-MP与HGPRT的亲和力。具体实施方式如下：
建立体外酶学反应，如图17A所示：
具体反应体系如下： 表7
2*Buffer 终浓度 体积(μl)
KCl(mM) 200 320
Tris 8.5(mM) 200 800
MgCl2(mM) 24 96
DTT(mM) 2 8
BSA 0.01 8
加入H2O   2776
总体积   4000
表8
Figure PCTCN2016073906-appb-000017
表9
次黄嘌呤 终浓度(μM) 6-MP 终浓度(μM)
1 250 1 1
2 125 2 0.5
3 62.5 3 0.25
4 31.25 4 0.125
5 15.625 5 0.0625
6 7.8125 6 0.03125
7 3.90625 7 0.015625
8 1.953125 8 0.0078125
9 0.9765625 9 0.00390625
10 0.48828125 10 0.001953125
11 0.244140625 11 0.000976563
次黄嘌呤与6-MP依次做浓度梯度稀释，37℃反应1小时，加入80％甲醇终止反应，通过LC-MS分别检测IMP与TIMP的含量。绘制反应曲线，通过Graphpad 5.0软件分别计算次黄嘌呤与6-MP的Km值。
实验结果如图17B所示，次黄嘌呤与HGPRT的亲和力明显高于6-MP与HGPRT的亲和力；且在100μM 6-MP作为反应底物时，随着加入次黄嘌呤浓度的升高，TIMP的生成受到了明显的抑制。
实施例19
洛美曲沙(Lometrexol)逆转PRPS1基因突变导致的6-MP药物耐受
洛美曲沙为一种GART的小分子抑制剂。本发明检测到洛美曲沙可以逆转PRPS1基因突变导致的6-MP药物耐受，具体步骤如下：
①细胞接种：将Reh-PRPS1-S103T与Reh-PRPS1-A190T细胞计数后接种到96孔板中，每孔接种104个细胞，每种细胞系设置5个复孔；
②细胞处理：用药物5ng/ml洛美曲沙(Lometrexol)或二甲亚枫(DMSO)对照预处理细胞1小时，对6-MP进行梯度稀释，加入铺好细胞的96孔板中，37℃培养72小时；
③读值：72小时后，每孔中加入50μl CellTiter-Glo试剂(Promega公司)，室温混匀孵育10分钟，放入酶标仪读化学发光值；
④计算：用Graphpad 5.0软件计算IC50值，比较差异；
⑤6-MP代谢产物浓度的检测：同实施例13；
⑥次黄嘌呤胞内浓度的检测：同实施例14。
实验结果如图18所示。与各自的对照相比，Reh-PRPS1-S103T与Reh-PRPS1-A190T细胞用洛美曲沙(Lometrexol)处理后药物6-MP的IC50值都显著下降，同时6-MP在体内的代谢产物TIMP与TGMP都显著上升，表明洛美曲沙使得Reh-PRPS1-S103T与Reh-PRPS1-A190T细胞系对6-MP的耐药性显著降低。并且处理后的Reh-PRPS1-S103T与Reh-PRPS1-A190T胞内的6-MP代谢产物TIMP与TGMP都有非常显著地上升，同时次黄嘌呤浓度有显著下降。
实施例20
质谱检测嘌呤代谢产物及嘌呤类似物药物代谢产物
药物及试剂
甲醇、乙腈(HPLC级)购自Sigma-Aldrich公司(美国)，甲酸(HPLC级)购自Merck公司(德国)，实验用水由Millipore-Q制备。
试验仪器
液质联用仪(UPLC-MS/MS)：
AB SCIEX
Figure PCTCN2016073906-appb-000018
5500(Singapore)
Waters Ultra Performance LC system(Singapore)
1.ACQUITYTMBinary Solvent Manager
2.ACQUITYTMColumn Manager
3.ACQUITYTMSample Organizer
4.ACQUITYTMSample Manager
采用Analyst,version 1.5.2数据采集和处理系统。
其他仪器：Thermo Fisher-70℃超低温冰箱(美国)；Eppendorf 5810R高速大容量低温离心机(德国)；IKA Vibrax VXR小型摇床(德国)；IKA Vortex振荡器(德国)；KQ5200DA超声波清洗器(昆山)等。
1.样品分析方法
样品处理方法
80％甲醇收集细胞，4度12000rpm离心5分钟，转移上清至新EP管中，取20μl进行LC-MS/MS分析。
1)标记的PRPP,ADP and GDP:
色谱条件
流动相组成：流动相A：50mM碳酸氢铵(pH9.5)
流动相B：乙腈:水＝6:1(v/v)
梯度洗脱：
表10
Figure PCTCN2016073906-appb-000019
Figure PCTCN2016073906-appb-000020
色谱柱：apHeraTM NH2Polymer(2×150mm)；
流速：0.6ml/min；进样体积：20μl；
质谱检测条件
采用电喷雾离子源(Turbo spray)，选择多通道反应监测(MRM)模式进行二级质谱分析。质谱检测工作参数及离子源参数如下：
表11
Figure PCTCN2016073906-appb-000021
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
色谱条件
流动相组成：流动相A：水-0.025％甲酸-1mM
流动相B：甲醇-0.025％甲酸-1mM醋酸铵
梯度洗脱：
表12
Figure PCTCN2016073906-appb-000022
色谱柱：Agilent Eclipse XDB-C18(4.6×150mm,5μm)；
流速：0.6ml/min；    进样体积：15μl；
质谱检测条件
采用电喷雾离子源(Turbo spray)，选择多通道反应监测(MRM)模式进行二级质谱分析。质谱检测工作参数及离子源参数如下：
表13
Figure PCTCN2016073906-appb-000023
实施例21
靶向嘌呤合成途径的核酸药物
针对嘌呤从头合成途径的慢病毒LV-shATIC,LV-shGART与LV-shPPAT的制备及其逆转PRPS1基因突变引起的6-MP药物耐受中的应用
①shRNA慢病毒载体的构建：针对ATIC,GART和PPAT的shRNA慢病毒载体GV298购自吉凯基因，用到的shRNA序列对应的DNA序列分别如下：shATIC-1 AATCTCTATCCCTTTGTAA:，shATIC-2:TGGAATCCTAGCTCGTAAT，shGART-1:CCAGGAGTTTGACTTACAA，shGART-2:CTAACTGTTGTCATGGCAA，shPPAT-1:CTTCGTTGTTGAAACACTT，shPPAT-2:TGTCTAACTGTAGACAAAT，shControl:TTCTCCGAACGTGTCACGT。
②将包装得到的对照慢病毒LV-shControl、慢病毒LV-shATIC1、LV-shATIC2、LV-shGART1、LV-shGART2、LV-shPPAT1与LV-shPPAT2。分别感染细胞系Reh、Reh-PRPS1-WT、Reh-PRPS1-S103T与Reh-PRPS1-A190T后形成稳定细胞系，感染Reh细胞的病毒MOI＝10，加入8μg/ml聚凝胺，感染24个小时后换液，培养48小时后加入0.8μg/ml嘌呤霉素抗性筛选一周后形成下述稳定细胞系：对照细胞系Reh-shControl、Reh-shATIC1、Reh-shATIC2、Reh-shGART1、Reh-shGART2、Reh-shPPAT1、Reh-shPPAT2、Reh-WT-shControl、Reh-WT-shATIC1、Reh-WT-shATIC2、Reh-WT-shGART1、Reh-WT-shGART2、Reh-WT-shPPAT1、Reh-WT-shPPAT2、Reh-S103T-shControl、Reh-S103T-shATIC1、Reh-S103T-shATIC2、Reh-S103T-shGART1、Reh-S103T-shGART2、Reh-S103T-shPPAT1、Reh-S103T-shPPAT2、Reh-A190T-shControl、Reh-A190T-shATIC1、Reh-A190T-shATIC2、Reh-A190T-shGART1、Reh-A190T-shGART2、Reh-A190T-shPPAT1和Reh-A190T-shPPAT2。
③细胞处理：将药物6-MP进行稀释，加入铺好如步骤②所述的几种细胞系的96孔板中，每种细胞系设置5个复孔，检测慢病毒感染细胞对6-MP的敏感性变化。具体实施方式同实施例8；
实验结果如图19所示，与空载病毒的对照细胞系Reh-S103T-shControl和Reh-A190T-shControl相比Reh-S103T-shATIC1、Reh-S103T-shATIC2、Reh-S103T-shGART1、Reh-S103T-shGART2、Reh-S103T-shPPAT1、Reh-S103T-shPPAT2和Reh-A190T-shATIC1、Reh-A190T-shATIC2、Reh-A190T-shGART1、Reh-A190T-shGART2、Reh-A190T-shPPAT1、Reh-A190T-shPPAT2的IC50值均下降，即表明对药物6-MP的耐药性降低。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (59)

  1. PRPS1作为急性淋巴细胞白血病耐药复发的基因标志物的用途。
  2. 一种PRPS1突变体，其特征在于，所述PRPS1突变体是氨基酸序列如序列表中SEQ ID No.2所示的序列中下述位点中的任意一个位点发生氨基酸的替换突变后形成的，所述替换为：第103位丝氨酸替换为苏氨酸、第103位丝氨酸替换为天冬酰胺、第144位天冬酰胺替换为丝氨酸、第176位赖氨酸替换为天冬酰胺、第183位天冬氨酸替换为谷氨酸、第190位丙氨酸替换为苏氨酸、第191位亮氨酸替换为苯丙氨酸、第303位苏氨酸替换为丝氨酸、第53位缬氨酸替换为丙氨酸、第72位异亮氨酸替换为缬氨酸、第77位半胱氨酸替换为丝氨酸、第139位天冬氨酸替换为甘氨酸、第311位酪氨酸替换为半胱氨酸、第103位丝氨酸替换为异亮氨酸、第114位天冬酰胺替换为天冬氨酸或第174位甘氨酸替换为谷氨酸。
  3. 一种PRPS1突变基因，其特征在于，所述PRPS1突变基因编码如权利要求2所述PRPS1突变体。
  4. 如权利要求3所述PRPS1突变基因，其特征在于，所述PRPS1突变基因的核苷酸序列为如序列表SEQ ID No.1所示的核苷酸序列中下述位点中的任意一个位点发生替换突变后形成，所述替换突变为：第308位G替换为C、第308位G替换为A、第431位A替换为G、第528位G替换为C、第549位C替换为G、第568位G替换为A、第573位G替换为C、第908位C替换为G、第158位T替换为C、第214位A替换为G、第230位G替换为C、第416位A替换为G、第932位A替换为G、第308位G替换为T、第340位A替换为G或第521位G替换为A。
  5. 一种重组载体，其特征在于，所述重组载体包含如权利要求3或4所述PRPS1突变基因。
  6. 一种转化体，其特征在于，所述转化体包含如权利要求5所述重组载体。
  7. 一组评估急性淋巴细胞白血病耐药复发风险的PRPS1突变体群，其特征在于，所述PRPS1突变体群包括以下PRPS1突变体：其氨基酸序列如序列表中SEQ ID No.2所示的序列中第103位丝氨酸替换为苏氨酸、第103位丝氨酸替换为天冬酰胺、第144位天冬酰胺替换为丝氨酸、第176位赖氨酸替换为天冬酰胺、第183位天冬氨酸替换为谷氨酸、第190位丙氨酸替换为苏氨酸、第191位亮氨酸替换为苯丙氨酸或第303位苏氨酸替换为丝氨酸。
  8. 如权利要求7所述PRPS1突变体群，其特征在于，所述PRPS1突变体群还包括以下PRPS1突变体中的一种或多种：其氨基酸序列如序列表中SEQ ID No.2所示的序列 中第53位缬氨酸替换为丙氨酸、第72位异亮氨酸替换为缬氨酸、第77位半胱氨酸替换为丝氨酸、第139位天冬氨酸替换为甘氨酸、第311位酪氨酸替换为半胱氨酸、第103位丝氨酸替换为异亮氨酸、第114位天冬酰胺替换为天冬氨酸、第174位甘氨酸替换为谷氨酸或第190位甘氨酸替换为缬氨酸。
  9. 一组评估急性淋巴细胞白血病耐药复发风险的PRPS1突变基因群，其特征在于，所述PRPS1突变基因群包括以下PRPS1突变基因：其核苷酸序列如序列表中SEQ ID No.1所示的序列中第308位G替换为C、第308位G替换为A、第431位A替换为G、第528位G替换为C、第549位C替换为G、第568位G替换为A、第573位G替换为C或第908位C替换为G。
  10. 如权利要求9所述PRPS1突变基因群，其特征在于，所述PRPS1突变基因群还包括以下PRPS1突变基因中的一种或多种：其核苷酸序列如序列表中SEQ ID No.1所示的序列中第158位T替换为C、第214位A替换为G、第230位G替换为C、第416位A替换为G、第932位A替换为G、第308位G替换为T、第340位A替换为G、第521位G替换为A或第569位C替换为T。
  11. 一种评估急性淋巴细胞白血病耐药复发风险的试剂盒，其特征在于，其包括：检测如权利要求9或10中所述PRPS1突变基因群中的PRPS1突变基因的试剂和使用说明书。
  12. 如权利要求11所述试剂盒，其特征在于，所述试剂包括扩增PRPS1基因各外显子的引物、DNA聚合酶、dNTP或缓冲液中的一种或多种。
  13. 如权利要求11或12所述试剂盒，其特征在于，所述使用说明书记载的内容包括以下步骤：
    (1)抽提样本基因组DNA；
    (2)检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因。
  14. 如权利要求13所述试剂盒，其特征在于，步骤(2)中，所述检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因的方法包括：以步骤(1)所得的样本基因组DNA为模板PCR扩增PRPS1基因各外显子，将扩增片段测序。
  15. 如权利要求13所述试剂盒，其特征在于，所述使用说明书记载的内容还包括步骤(3)：按以下标准评估急性淋巴细胞白血病耐药复发的风险：检测到样本基因组DNA中存在如权利要求9或10所述PRPS1突变基因群中的任意一种或多种PRPS1突变基因，则该样本有急性淋巴细胞白血病耐药复发的风险。
  16. 如权利要求12所述试剂盒，其特征在于，所述扩增PRPS1基因各外显子的引 物为核苷酸序列如序列表中SEQ ID NO.3～SEQ ID NO.16所示序列中的一种或多种；和/或，所述试剂盒包括提取细胞或组织样本DNA的试剂；和/或，所述抽提样本基因组DNA的试剂为蛋白酶、饱和酚、体积比为24：1的氯仿和异戊醇混合液、醋酸钠、无水乙醇、70％乙醇和TE溶液，所述百分比为体积百分比；和/或，所述提取细胞或组织样本DNA的试剂为Qiagen公司生产的DNA抽提试剂盒；和/或，所述DNA聚合酶为TOYOBO公司生产的KOD-Plus DNA聚合酶；和/或，所述缓冲液为TOYOBO公司生产的KOD-Plus DNA聚合酶缓冲液；和/或，所述dNTP为dATP、dGTP、dCTP和dTTP四种的混合物。
  17. 一种评估急性淋巴细胞白血病耐药复发风险的试剂盒，其特征在于，其包括：检测如权利要求7或8中所述PRPS1突变体群中的PRPS1突变体的试剂和使用说明书。
  18. 如权利要求17所述试剂盒，其特征在于，所述试剂为检测PRPS1突变体酶活性的试剂。
  19. 如权利要求18所述试剂盒，其特征在于，所述试剂包括PRPP和用同位素碳原子标记的葡萄糖。
  20. 如权利要求19所述试剂盒，其特征在于，所述用同位素碳原子标记的葡萄糖为13C标记的葡萄糖。
  21. 如权利要求17-20中任一项所述试剂盒，其特征在于，所述使用说明书记载的内容包括以下步骤：
    (1)裂解样本细胞；
    (2)检测步骤(1)所得的细胞裂解液中的PRPS1突变体。
  22. 如权利要求21所述试剂盒，其特征在于，所述使用说明书记载的内容还包括步骤(3)：检测到样本中存在酶活性高于野生型PRPS1的PRPS1突变体，则该样本有急性淋巴细胞白血病耐药复发的风险；所述酶活性高于野生型PRPS1的PRPS1突变体较佳地为如权利要求7或8所述PRPS1突变体群中的任意一种或多种。
  23. 如权利要求21所述试剂盒，其特征在于，步骤(2)所述检测步骤(1)所得的细胞裂解液中的PRPS1突变体的方法是检测PRPS1的酶活性。
  24. 如权利要求17-20中任一项所述试剂盒，其特征在于，所述试剂盒还包括细胞裂解试剂，所述细胞裂解试剂较佳地为80％甲醇，所述百分比为体积百分比；和/或，所述试剂盒还包括无葡萄糖培养基，所述无葡萄糖培养基较佳地为购自Gibco公司的无葡萄糖培养基。
  25. 一种检测化合物的治疗或预防儿童急性淋巴细胞白血病耐药复发的活性的方法，其特征在于，其包括下述步骤：
    (1)使化合物与含如权利要求9或10所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，得预处理的白血病细胞；
    (2)使抗白血病的化疗药物与步骤(1)所得的预处理的白血病细胞接触。
  26. 一种筛选治疗或预防儿童急性淋巴细胞白血病耐药复发的药物的方法，其特征在于，其包括下述步骤：
    (1)使候选化合物与含如权利要求9或10所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，得预处理的白血病细胞；
    (2)使抗白血病的化疗药物与步骤(1)所得的预处理的白血病细胞接触。
  27. 如权利要求26所述方法，其特征在于，步骤(1)所述白血病细胞为人源白血病细胞，所述人源白血病细胞较佳地为人源淋巴白血病细胞，更佳地为Reh细胞或Jurkat细胞。
  28. 如权利要求26所述方法，其特征在于，所述接触的方法包括将候选化合物直接添加到所述白血病细胞的培养基中；和/或，步骤(1)中还同时不采用候选化合物与含如权利要求9或10所述PRPS1突变基因群中的任何一种PRPS1突变基因的白血病细胞接触，作为对照；和/或，步骤(2)中所述化疗药物为嘌呤类药物，所述嘌呤类药物较佳地为6-MP或6-TG。
  29. 如权利要求26所述方法，其特征在于，还包括步骤(3)，即检测步骤(2)所得的细胞的存活率，所述检测的方法较佳地为使用活细胞染料染色后计数法、MTT法或通过测量荧光素酶与存活细胞内的ATP结合后发出荧光强度的方法，所述通过测量荧光素酶与存活细胞内的ATP结合后发出荧光强度的方法较佳地为使用市售的Promega公司的Cell Titer-Glo试剂来检测。
  30. 一种预防或治疗急性淋巴细胞白血病耐药复发的药物的靶标，其特征在于，所述靶标为嘌呤从头合成途径中所有酶中的一种或多种。
  31. 如权利要求30所述靶标，其特征在于，所述靶标为选自PRPS1、PRPS2、PPAT、PFAS、GART、ATIC、PAICS和ADSL中的一种或多种。
  32. 一种siRNA，其特征在于，所述的siRNA包括正义RNA片段与反义RNA片段，所述正义RNA片段包含如权利要求1所述靶标的DNA编码的RNA分子，所述正义RNA片段与反义RNA片段能够互补形成双链RNA分子。
  33. 一种向导RNA，其特征在于，其核苷酸序列如SEQ.ID No.41所示。
  34. 一种慢病毒载体，其特征在于，其含有如权利要求33所述向导RNA的DNA序列和Cas9基因。
  35. 一种慢病毒，其特征在于，其由如权利要求34所述慢病毒载体在慢病毒包装质粒的辅助下在细胞系中经病毒包装而成。
  36. 一种向导RNA，其特征在于，其核苷酸序列如SEQ.ID No.42所示。
  37. 一种慢病毒载体，其特征在于，其含有如权利要求36所述向导RNA的DNA序列和Cas9基因。
  38. 一种慢病毒，其特征在于，其由如权利要求37所述慢病毒载体在慢病毒包装质粒的辅助下在细胞系中经病毒包装而成。
  39. 如权利要求34和37所述慢病毒载体，其特征在于，所述Cas9基因为按照人类遗传密码子优化后的Cas9基因。
  40. 一种如权利要求33和/或36所述向导RNA、如权利要求34和/或37所述慢病毒载体或如权利要求35和/或38所述慢病毒在制备治疗或预防急性淋巴细胞白血病耐药复发的药物中的应用。
  41. 一种治疗或预防急性淋巴细胞白血病耐药复发的药物组合物，其特征在于，其有效物质含有选自如权利要求32所述siRNA、如权利要求33和/或36所述向导RNA、如权利要求34和/或37所述慢病毒载体和如权利要求35和/或38所述慢病毒中的一种或多种。
  42. 一种嘌呤从头合成途径中所有酶中的一种或多种酶的抑制剂在制备预防或治疗急性淋巴细胞白血病耐药复发的药物中的应用。
  43. 如权利要求42所述的应用，其特征在于，所述抑制剂为抑制性RNA、抑制性多肽、抗体和小分子化合物抑制剂中的一种或多种。
  44. 如权利要求43所述应用，其特征在于，所述抑制性RNA为选自向导RNA、siRNA、shRNA、miRNA、反义RNA和核酶中的一种或多种。
  45. 如权利要求43所述应用，其特征在于，所述小分子化合物抑制剂为选自洛美曲沙和力比泰中的一种或两种。
  46. 一种评估急性淋巴细胞白血病耐药复发风险的试剂盒，其特征在于，其包括裂解样本细胞的试剂、检测次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的试剂和使用说明书；所述使用说明书记载的内容包括以下步骤：
    (a)收集、裂解样本细胞；
    (b)检测步骤(a)所得的样本细胞裂解液中的选自次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的含量；
    (c)通过步骤(b)所得的次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的浓度判断样本急性淋巴细胞白血病耐药复发的风险。
  47. 如权利要求46所述试剂盒，其特征在于，所述试剂盒还包括标准品，所述标准品为次黄嘌呤、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中的一种或多种。
  48. 如权利要求47所述试剂盒，其特征在于，所述标准品为5－氨基咪唑－4－甲酰胺核苷酸、次黄苷和全部碳原子为13C、全部氮原子为15N的次黄嘌呤中的一种或多种。
  49. 如权利要求46-48中任一项所述试剂盒，其特征在于，所述裂解样本细胞的试剂为80％甲醇，所述百分比为体积百分比。
  50. 如权利要求46-48中任一项所述试剂盒，其特征在于，所述检测次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中的一种或多种的试剂为质谱检测试剂。
  51. 一种评估病人ALL耐药复发风险的检测方法，其特征在于，所述方法包括以下步骤：
    (1)抽提样本基因组DNA；
    (2)检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因；
  52. 如权利要求51所述方法，其特征在于，所述方法还包括步骤(3)：按以下标准评估急性淋巴细胞白血病耐药复发的风险：检测到样本基因组DNA中存在如权利要求9或10所述PRPS1突变基因群中的任意一种或多种PRPS1突变基因，则该样本有急性淋巴细胞白血病耐药复发的风险。
  53. 如权利要求51或52所述方法，其特征在于，步骤(1)中，所述样本为待检病人的骨髓来源或外周血来源的肿瘤细胞或体外培养的永生化细胞系；和/或，步骤(2)中，所述检测步骤(1)所得的样本基因组DNA中的PRPS1突变基因的方法包括：以步骤(1)所得的样本基因组DNA为模板PCR扩增PRPS1基因各外显子，将扩增片段测序。
  54. 一种通过检测PRPS1酶活性来评估儿童ALL耐药复发风险的方法，其特征在于，所述方法包括以下步骤：
    (1)裂解样本细胞；
    (2)检测步骤(1)所得的细胞裂解液中的PRPS1突变体。
  55. 如权利要求54所述方法，其特征在于，所述步骤(1)包括：(a)分离ALL病人的白血病细胞；(b)培养步骤(a)中所述白血病细胞；(c)用化疗药物处理步骤(b) 所述白血病细胞；(d)收集步骤(c)所述白血病细胞，裂解；和/或，所述步骤(2)包括：(e)通过LC-MS检测PRPP含量。
  56. 如权利要求54所述方法，其特征在于，步骤(c)中，所述化疗药物为嘌呤类似物类化疗药物，较佳地为6-MP或6-TG。
  57. 一种评估急性淋巴细胞白血病耐药复发风险的方法，其特征在于，所述方法包括以下步骤：
    (a)收集、裂解样本细胞；
    (b)检测步骤(a)所得的样本细胞裂解液中的选自次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的含量；
    (c)通过步骤(b)所得的次黄嘌呤、次黄嘌呤核苷酸单磷酸、5－氨基咪唑－4－甲酰胺核苷酸和次黄苷中一种或多种的浓度判断样本急性淋巴细胞白血病耐药复发的风险。
  58. 如权利要求57所述方法，其特征在于，步骤(a)中所述样本细胞为含有如权利要求7或8中所述PRPS1突变体的急性淋巴细胞白血病样本细胞；和/或，步骤(a)中使用80％甲醇裂解所述样本细胞，所述百分比为体积百分比；和/或，步骤(b)中所述检测为质谱检测。
  59. 如权利要求58所述方法，其特征在于，步骤(a)中所述样本细胞为含有第103位丝氨酸替换为苏氨酸或第190位丙氨酸替换为苏氨酸的PRPS1突变体的Reh细胞。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009084753A1 (en) * 2007-12-28 2009-07-09 Sungkyunkwan University Foundation For Corporate Collaboration Mutation of prps1 gene causing cmtx5 disease and the use thereof
WO2014116704A1 (en) * 2013-01-22 2014-07-31 The Regents Of The University Of California Phosphoribosyl pyrophosphate synthetase 2 (prps2) as a therapeutic target in cancer treatment

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2662508A1 (en) 2006-09-19 2008-07-10 Novartis Ag Biomarkers of target modulation, efficacy, diagnosis and/or prognosis for raf inhibitors
MX2010013647A (es) * 2008-06-12 2011-04-05 Affiris Ag Compuestos para tratar sintomas asociados con la enfermedad de parkinson.
CN105986023B (zh) 2015-02-17 2021-01-05 上海交通大学医学院附属上海儿童医学中心 急性淋巴细胞白血病耐药复发相关突变基因及其应用
CN105985960B (zh) 2015-02-17 2019-12-06 上海交通大学医学院附属上海儿童医学中心 治疗或预防急性淋巴细胞白血病耐药复发的药物及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009084753A1 (en) * 2007-12-28 2009-07-09 Sungkyunkwan University Foundation For Corporate Collaboration Mutation of prps1 gene causing cmtx5 disease and the use thereof
WO2014116704A1 (en) * 2013-01-22 2014-07-31 The Regents Of The University Of California Phosphoribosyl pyrophosphate synthetase 2 (prps2) as a therapeutic target in cancer treatment

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LI, BENSHANG ET AL.: "Negative Feedback-defective PRPS1 Mutants Drive Thiopurine Resistance in Relapsed Childhood ALL", NATURE MEDICINE, vol. 21, no. 6, 30 June 2015 (2015-06-30), pages 563 - 573 *
LI, JIANZHONG ET AL.: "Mutations in PRPS1 and Hereditary Hearing Loss", CHINESE JOURNAL OF OTOLOGY, vol. 8, no. 1, 31 December 2010 (2010-12-31), pages 57 - 62 *
LI, ZHONGQIN ET AL.: "Catalytic Spectrophotometric Quantitation for Hypoxanthine by Conjugating Xanthine Oxidase with Horseradish Peroxidase", SPECTROSCOPY AND SPECTRAL ANALYSIS, vol. 28, no. 9, 30 September 2008 (2008-09-30), pages 2169 - 2172 *
MULLIGHAN, C.G.;: "Mutant PRPS1: a New Therapeutic Target in Relapsed Acute Lymphoblastic Leukemia", NATURE MEDICINE, vol. 21, no. 6, 30 June 2015 (2015-06-30), pages 553 - 554 *

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