A novel target for the treatment of philadelphia chromosome-positive leukemia and application thereof

Abstract

The present application relates to the field of biological medicine, in particular to the preparation of a drug or diagnostic reagent for treating Philadelphia chromosome positive leukemia by taking synaptic receptor associated protein RAPSYN or its gene as a target point. The present application specifically discloses the use of RAPSYN gene or protein as a therapeutic target in screening and / or preparation of a drug for preventing and / or treating Philadelphia chromosome positive leukemia. Philadelphia chromosome positive leukemia can be treated by negatively regulating the level and activity of RAPSYN gene or protein. The efficacy and prognosis of patients can be predicted by detecting the expression level of RAPSYN. The treatment strategy or drug taking RAPSYN gene and protein as a target point can be used as a new means of leukemia targeted therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and particularly relates to the discovery of RAPSYN as a target for treating Philadelphia chromosome positive leukemia and application thereof. BACKGROUND

[0002] Philadelphia chromosome (Ph) is formed by translocation between chromosome 22 and chromosome 9, which leads to the fusion of BCR and ABL genes. The fusion gene is transcribed and translated into BCR-ABL protein with persistent tyrosine kinase activity. The protein can phosphorylate downstream interacting proteins through autophosphorylation, thereby activating signaling pathways and promoting the development of leukemia (Chereda, B. and Melo, J., Ann Hematol., 2015, 94:S107-S121). The BCR-ABL fusion gene exists in all Philadelphia chromosome-positive leukemia patients, which can be used as a biological diagnostic marker, efficacy monitoring and biological target for drug targeted therapy. The first-line drugs for treating leukemia are mostly inhibitors of BCR-ABL1 protein kinase, such as Imatinib, which has made important contributions to the treatment of Philadelphia chromosome-positive leukemia. However, with the popularization of clinical use, drug resistance has become increasingly prominent. Point mutations in the BCR-ABL kinase domain are the main cause of drug resistance or recurrence of tyrosine kinase inhibitors in patients. These mutations lead to amino acid substitution in the BCR-ABL1 protein kinase domain, which disrupts the binding between Imatinib and BCR-ABL1 protein, directly or indirectly causing decreased drug sensitivity in patients. To overcome the drug resistance of Imatinib, Dasatinib, Nilotinib and other second-generation BCR-ABL1 protein kinase inhibitors have been introduced, which can overcome almost all drug-resistant leukemias caused by kinase mutations except T315I mutation. Although the inhibitor Ponatinb, which can treat T315I mutation-resistant leukemia, was introduced in 2012, it is powerless for some mixed mutations such as E255V / T315I double mutation (Flis S. and Chojnacki, T., Drug Design Develop. Ther., 2019, 13:825-843). Currently, cytogenetics and molecular biology are mainly used to monitor the efficacy of TKIs, and the detection and monitoring of BCR-ABL mRNA expression level has certain limitations. Screening of differential genes between normal cells and Philadelphia chromosome-positive leukemia cells, and using the expression level of differential genes as a monitor and diagnostic marker for leukemia progression and differential genes as a drug target for the development or screening of drugs for the treatment of Philadelphia chromosome-positive leukemia is a new research direction for the treatment of drug-resistant leukemia. Therefore, it is urgent to find new targets for the treatment of drug-resistant leukemia other than BCR-ABL kinase.

[0003] Protein neddylation modification is a post-translational modification process, which mainly covalently binds small molecule protein NEDD8 to lysine residues of substrate proteins through the process of E1-E2-E3 enzymatic reaction. The classic substrate of neddylation modification is the cullin family protein, and its specific E3 ligase is the cullin-RING E3 ligase (CRL) (Radoslav I. et al., Nat. Rev. Mol. Cell Biol., 2015, 16: 30-44.). The dysfunction of CRL can lead to tumorigenesis and promote tumor development. In addition, recent studies have shown that NEDD8 can modify many tumor-related proteins, such as p53, MDM2 and Smurf, etc. More importantly, in many tumors, the entire process of neddylation modification, including E1, E2, E3 and modified substrates, is over-activated during tumor development. In addition, the biological function of neddylation modification has substrate specificity, indicating that neddylation modification plays an important role in the occurrence and development of tumors and can be used as a potential target for the treatment of tumors. Clarifying whether the BCR-ABL fusion protein is post-translationally modified can regulate it at the protein level rather than at the kinase activity level, and finding specific E3 ligases that mediate the post-translational modification of BCR-ABL protein has important application value for the treatment of drug-resistant or recurrent leukemia.

[0004] Synapse-associated protein RAPSYN is a 43 kDa protein, whose primary structure can be divided into three parts, respectively, seven tetratricopeptide repeat (TRP) composed of amino acids in 1-287 segments, coiled coil domain (CCD) composed of amino acids in 288-348 segments and zinc-finger domain (ZnF) composed of amino acids in 363-348 segments (Antolik C. et al., Neuroscience, 2006, 141: 87-100.). The functional study of these regions is mainly focused on the influence on the formation and aggregation of acetylcholine receptor (AChR). At the same time, RAPSYN protein contains a special RING region domain. In view of the fact that many E3 ligases contain RING region, Mei. Li. group found that the RING region mutant of RAPSYN has E3 ligase activity and can mediate neddylation modification of the substrate when the RING region mutant and non-mutant of RAPSYN were detected for E3 ligase catalytic activity in vitro (Li et al., Neuron, 2016, 92: 1007-1019). So far, there is no report on the application of RAPSYN as a target for treating leukemia patients. In view of the current limitations, the present application first found that RAPSYN gene is highly expressed in Philadelphia chromosome positive leukemia cells. Based on the difference in expression between normal cells and leukemia cells, it is first clarified that RAPSYN can be a new and specific leukemia-related gene and can become a new generation of drug development target. SUMMARY

[0005] BCR-ABL has been used as a target for treating Philadelphia chromosome positive leukemia, and the corresponding inhibitors have been updated, but with the popularization of clinical use, drug resistance problems have become increasingly prominent. In order to solve the above problems existing in the prior art, the application provides an application of RAPSYN gene and protein as a target for treating Philadelphia chromosome positive leukemia. It is found in the research that the expression level of RAPSYN gene and protein in Philadelphia chromosome positive leukemia is significantly increased, and it is found that the expression level of the gene is related to the prognosis of Philadelphia chromosome positive leukemia, which indicates that RAPSYN can be used as a new Philadelphia chromosome positive leukemia regulator. The discovery of the gene not only provides a new theoretical basis for the development mechanism of Philadelphia chromosome positive leukemia, but more importantly, it also provides a new way for the development of Philadelphia chromosome positive leukemia drugs. Taking RAPSYN gene and protein as a target, new Philadelphia chromosome positive leukemia treatment drugs can be further screened, at the same time, RAPSYN can also be used as a detection index to evaluate the drug efficacy of Philadelphia chromosome positive leukemia and applied to the preparation of Philadelphia chromosome positive leukemia prediction, detection or prognosis judgment kit.

[0006] The application provides that the synaptic receptor associated protein RAPSYN is used as a target in the development or screening of drugs or gene products for treating Philadelphia chromosome positive leukemia, and the gene sequence of the synaptic receptor associated protein RAPSYN is gene ID: 5913 in NCBI. The synaptic receptor associated protein amino acid sequence NM_005055.5 is specifically shown in SEQ ID NO. 1.

[0007] The drug or gene product has at least one of the following functions (1)-(6): (1) reducing the expression of BCR-ABL; (2) prolonging the survival period of Philadelphia chromosome positive leukemia patients; (3) improving the clinical symptoms of Philadelphia chromosome positive leukemia patients; (4) increasing the sensitivity of Philadelphia chromosome positive leukemia patients to tyrosine kinase inhibitor treatment; (5) reducing the number of leukemia stem cells; (6) used for combined use and treatment with tyrosine kinase inhibitors.

[0008] Further, the drug is a negative regulator of RAPSYN protein or its encoding gene. The negative regulator includes a protein inhibitor, a nucleic acid aptamer and / or an interfering RNA, a gRNA, a microRNA, a small molecule compound inhibitor of RAPSYN protein and a combination of the above inhibitors.

[0009] Preferably, the negative regulator is selected from double-stranded siRNA and / or shRNA based on a carrier of the RAPSYN encoding gene.

[0010] In one specific example of the present application, the nucleotide sequence of the target sequence combined with the shRNA is shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0011] Preferably, the negative regulator is selected from a microRNA of a RAPSYN-encoding gene.

[0012] The drug, small molecule compound inhibitor, protein inhibitor, and nucleic acid aptamer or inhibitor of the combination thereof of the present application can target the transcription or expression of the RAPSYN gene or target the E3 ligase activity of the RAPSYN protein to inhibit the proliferation of Philadelphia chromosome-positive leukemia cells, thereby achieving targeted inhibition of the occurrence and development of Philadelphia chromosome-positive leukemia.

[0013] In one specific example of the present application, the reduction of the expression of RAPSYN can inhibit the proliferation of Philadelphia chromosome-positive leukemia cells, reduce the clonogenicity and tumorigenicity of Philadelphia chromosome-positive leukemia cells, and prolong the survival of Philadelphia chromosome-positive mice.

[0014] The drug or gene product can inhibit neddylation modification and other ubiquitination modification of proteins such as BCR-ABL, SET, Hsp90, and SAM68, can accelerate the degradation of BCR-ABL protein by targeting RAPSYN, and inhibit the proportion of leukemia stem cells.

[0015] Further, the drug of the present application further comprises a tyrosine kinase inhibitor.

[0016] The present application also discloses the use of the synaptic receptor-associated protein RAPSYN or its gene in the preparation of a diagnostic reagent for Philadelphia chromosome-positive leukemia.

[0017] The use is to prepare a Philadelphia chromosome-positive leukemia prediction, detection, or prognosis judgment reagent by taking RAPSYN or its gene as a detection target.

[0018] The diagnostic reagent contains a primer sequence that specifically amplifies the synaptic receptor-associated protein RAPSYN gene.

[0019] The detection reagent kit contains a reagent for detecting the expression amount of RAPSYN. By detecting the expression level of RAPSYN in the sample, the prediction, detection, or prognosis judgment of Philadelphia chromosome-positive leukemia is achieved.

[0020] In the use, the drug or gene product can target the transcription or expression of the RAPSYN gene.

[0021] In the application, the drug or gene product can target to inhibit the E3 ligase activity of RAPSYN protein.

[0022] In the application, the drug or gene product can target to inhibit the proliferation of Philadelphia chromosome positive leukemia cells.

[0023] In the application, the drug or gene product can target to inhibit the tumorigenic ability of Philadelphia chromosome positive leukemia cells, prolong the survival of leukemia mice.

[0024] In the application, the drug or gene product can target to inhibit neddylation modification of proteins such as BCR-ABL, SET, Hsp90, SAM68, etc.

[0025] In the application, the drug or gene product can target to accelerate the degradation of BCR-ABL protein.

[0026] In the application, the drug or gene product can target to inhibit the proportion of leukemia stem cells.

[0027] The effective effect of the present application is that:

[0028] The present application first discloses the correlation between RAPSYN and the occurrence and development of Philadelphia chromosome positive leukemia. The research results of the present application show that the expression level of RAPSYN in Philadelphia chromosome positive cell lines is significantly increased. In addition, RAPSYN mediates neddylation modification of BCR-ABL protein as an E3 ligase, thereby increasing the stability of the protein and promoting the development of Philadelphia chromosome positive leukemia. The present application proves that reducing the expression of RAPSYN can significantly inhibit the proliferation of Philadelphia chromosome positive leukemia cells, reduce the proportion of leukemia stem cells, slow down the tumor growth and prolong the survival of Philadelphia chromosome positive leukemia mice. Based on the above research, the present application proposes that RAPSYN can be used as a new therapeutic target for Philadelphia chromosome positive leukemia in Philadelphia chromosome positive leukemia cells, which has important application value for the development and screening of Philadelphia chromosome positive leukemia treatment drugs. In addition, RAPSYN is an important oncogene, and based on the difference in its expression in Philadelphia chromosome positive leukemia cells and normal bone marrow stromal cells, it can be used as a detection target for Philadelphia chromosome positive leukemia prediction and Philadelphia chromosome positive leukemia treatment effect monitoring. BRIEF DESCRIPTION OF DRAWINGS

[0029] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0030] Figure 1 Expression of RAPSYN gene (A) and protein (B) in normal bone marrow stromal cell HS-5, Philadelphia chromosome positive leukemia cell K562, KU812 and MEG-01.

[0031] Figure 2 mRNA expression level (A) and protein expression level (B) of RAPSYN after transfection of Philadelphia chromosome positive leukemia cell K562, KU812 and MEG-01 with lentivirus packaged shRNA of RAPSYN.

[0032] Figure 3 Inhibition of growth of K562 cell after silencing expression of RAPSYN by shRNA of RAPSYN.

[0033] Figure 4 Inhibition of growth of MEG-01 cell after silencing expression of RAPSYN by shRNA of RAPSYN.

[0034] Figure 5 Inhibition of growth of KU812 cell after silencing expression of RAPSYN by shRNA of RAPSYN.

[0035] Figure 6 Knockout of RAPSYN inhibits development of leukemia. (A figure is protein level of RAPSYN in cell after knockout of RAPSYN; B figure is inhibition of K562 cell clone formation by knockout of RAPSYN; C figure is slowing of tumor formation in mouse by knockout of RAPSYN; D figure is prolongation of survival period of Philadelphia chromosome positive leukemia mouse by knockout of RAPSYN).

[0036] Figure 7 RAPSYN as E3 ligase mediates neddylation modification of BCR-ABL (A figure is promotion of neddylation modification of BCR-ABL by NEDD8; B figure is change of modification level of BCR-ABL detected in RAPSYN knockout cell line and wild type cell).

[0037] Figure 8 Change of half-life of BCR-ABL protein after knockout of RAPSYN.

[0038] Figure 9 Change of proportion of LSK stem cell in bone marrow of mouse after knockout of RAPSYN. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with the drawings and specific examples.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0041] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified, for example: cells: K562, KU812 and MEG-01 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. HS-5 cells were purchased from the American ATCC cell bank.

[0042] Antibodies: BCR-ABL, RAPSYN antibodies were purchased from Abeam, USA; β-Tubulin, NEDD8 and secondary antibody were purchased from Cell Signaling Technology Co., Ltd., USA.

[0043] Reagents: 1640 medium, DMEM high-sugar medium were purchased from Nanjing Kanghao Biotechnology Co., Ltd.

[0044] Real-time fluorescent quantitative PCR (RT-PCR): After the cells grow to an appropriate number, the culture medium is discarded and washed with PBS for 2 times, and the total RNA of the cells is extracted by Trizol method. After determining the RNA concentration, the RNA is reversely transcribed into cDNA. The primers corresponding to the detection genes are designed, and the expression of the corresponding genes is detected by SYBR Green I method.

[0045] Western blot analysis: After the cells grow to an appropriate number, the culture medium is discarded and washed with PBS for 2 times, and NP40 cell lysis solution is added, and lysis for 30 minutes on ice, centrifuged at 4℃ 12000 rpm for 15 minutes, and the supernatant is collected. The cell concentration is detected by BCA kit, and the protein lysis solution concentration is adjusted, and the corresponding 5x protein loading buffer is added, boiled for 5 minutes, and stored at -20℃ for standby. The protein sample is separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. 5% blocking solution is used for blocking at room temperature for 2 hours, and then incubated with the corresponding primary antibody at 4℃ overnight. Then, the membrane is incubated with the corresponding secondary antibody at room temperature for 2 hours, and analyzed by infrared imaging system.

[0046] Co-immunoprecipitation analysis: 1 mg of lysed cell protein sample was incubated with 10 μg of antibody at 4℃ overnight. 40 μL of protein A / G agrose was aspirated into a 1.5 mL centrifuge tube, and washed twice with cell lysis solution. The protein lysis solution and antibody co-incubated sample was added to the centrifuge tube, and incubated at 4℃ with gentle rotation for 3 hours. After incubation, wash twice with cell lysis solution, then add 60 μL of loading buffer to resuspend the protein A / G agrose, prepare the sample at 100℃, and perform Western blot detection.

[0047] Experimental animals: The NCG mice used in this example were purchased from Jiangsu Jizu Biological Technology Co., Ltd.

[0048] Data analysis: Data analysis was performed using GraphPad Prism software. The mean ± standard deviation (X ± SD) was used to represent the measurement data, and Student's t test was used to analyze the differences between the two groups. P<0.05 was considered statistically significant. The experiment was repeated at least three times.

[0049] Example 1: Expression of RAPSYN gene and protein in leukemia

[0050] In order to detect the expression of RAPSYN gene and protein in leukemia cells, the expression of RAPSYN in normal bone marrow stromal cells HS-5 and Philadelphia chromosome positive leukemia cell lines K562, KU812 and MEG-01 cells was detected.

[0051] First, the expression level of RAPSYN mRNA in four kinds of cells was detected by real-time fluorescence quantitative PCR (RT-PCR): four kinds of cells were collected, washed twice with pre-cooled PBS, and 1 mL Trizol reagent was added. 4℃ for 15 minutes, so that the cells were fully lysed. Then chloroform and isopropanol were added to extract RNA, and the concentration was determined. The extracted mRNA was reverse transcribed into cDNA using a reverse transcription kit. RAPSYN specific primers were designed, and AceQ Qpcr SYBR Green Master Mix kit was used to detect the expression of RAPSYN mRNA in four kinds of cells.

[0052] Secondly, the expression level of RAPSYN protein in four kinds of cells was detected by Western blot analysis: four kinds of cells were collected, the culture medium was discarded and washed with PBS for 2 times, NP40 cell lysis buffer was added, and lysis was carried out on ice for 30 minutes. 4℃ 12000rpm centrifugation for 15 minutes, collect supernatant. The cell concentration was detected by BCA kit, and the protein lysis buffer concentration was adjusted, then the corresponding 5X protein loading buffer was added, boiled in boiling water for 5 minutes, and stored at -20℃ for standby. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. 5% blocking solution was used for blocking at room temperature for 2 hours, and then incubated with the corresponding primary antibody at 4℃ overnight. Then, the membrane was incubated with the corresponding secondary antibody at room temperature for 2 hours, and analyzed by infrared imaging system.

[0053] The results are shown in Figure 1 A and Figure 1 B, Figure 1A shows that RAPSYN gene is significantly highly expressed in Philadelphia chromosome positive leukemia cells K562, KU812 and MEG-01 compared with normal bone marrow stromal cells HS-5. Figure 1 B shows that RAPSYN protein is significantly highly expressed in Philadelphia chromosome positive leukemia K562, KU812 and MEG-01 cells compared with normal cells HS-5. The results show that the expression of RAPSYN gene and protein in Philadelphia chromosome positive leukemia cells is significantly increased compared with normal cells HS-5.

[0054] Example 2: shRNA targeting RAPSYN gene inhibits the growth of Philadelphia chromosome positive leukemia cells According to the sequence of RAPSYN gene, specific shRNA (target sequence is SEQ ID NO: 2-4) is designed to silence the expression of RAPSYN, and is packaged into lentivirus to transfect three Philadelphia chromosome positive leukemia cells K562, KU812 and MEG-01.

[0055] Among them, the method of lentivirus packaging is as follows: first, the designed three shRAPSYN (target sequence is SEQ ID NO: 2-4, numbered #1-#3 in turn) sequences are cloned into pLKO-EGFP-puro vector. The plasmid pLKO-EGFP containing shRAPSYN sequence or the corresponding empty vector is co-transfected with virus packaging plasmid (pLP1, pLP2, pLP / VSVG) into HEK293T cells. After 6-8 hours of transfection, fresh culture and replacement of transfection reagent are used. After 72 hours of continuous culture, the produced lentivirus supernatant is collected and filtered with 0.22 μM filter. 15 mL filtered lentivirus supernatant is concentrated by 100 KD ultrafiltration tube at 2000 rpm, 4℃ for 1 hour, and the lentivirus concentrate is stored at -80℃.

[0056] The method of lentivirus transfection of shRNA is as follows: 0.5×10 5 Cells are inoculated into 35 mm cell culture dishes, 2 mL of 1640 cell culture medium is added to each dish, and the dishes are cultured in a cell culture incubator overnight. When the density of the cells reaches more than 60%, the cells are infected with lentivirus containing 5 μL of shRNA. After 48 hours of infection, the infected positive cells are screened with 1 μg / mL of puromycin drug, so as to obtain a cell strain stably expressing shRNA, and finally the transfection efficiency is detected by real-time fluorescent quantitative PCR and immunoblotting technology. As shown in Figure 2 A, the results of real-time fluorescent quantitative PCR show that the mRNA expression level of RAPSYN in the three shRNA treatment groups is significantly reduced. Consistently, the results of immunoblotting show that the protein expression level of RAPSYN in the three shRNA treatment groups is significantly reduced, as shown inFigure 2 B.

[0057] Further, the effect of the above shRNA silencing RAPSYN expression on the proliferation of three Philadelphia chromosome-positive leukemia K562, KU812 and MEG-01 cells was detected by a competitive growth experiment.

[0058] The method of the competitive growth experiment is as follows: In order to detect the toxicity of shRAPSYN, the lentiviral vector used carries a GFP fluorescent tag to indicate the proportion of infected cells. After transfecting the three cells with shRAPSYN for two days, 5000 cells were taken for flow cytometry detection to determine the initial proportion of GFP-positive living cells. Then, sampling was taken every two days. The GFP-positive cells at each time point were compared with the positive cells of the next day to determine the change in the cell proliferation ability after reducing the expression of RAPSYN.

[0059] The results are shown in Figure 3 , Figure 4 and Figure 5 . The results show that inhibiting the expression of RAPSYN can significantly inhibit the proliferation of cells.

[0060] Example 3: CRISPR-Cas9 knockout of RAPSYN gene inhibits the occurrence and development of Philadelphia chromosome-positive leukemia Jiangsu Jikai Biotechnology Co., Ltd. was commissioned to knockout the RAPSYN gene in K562 cells using CRISPR-Cas9 technology to obtain a RAPSYN-knockout K562 cell line. First, the efficiency of RAPSYN knockout and its effect on BCR-ABL protein were detected by immunoblotting. Second, the cell clonogenicity and tumorigenicity after knocking out RAPSYN were detected.

[0061] Cell clonogenicity experiment: 1.2% and 0.6% low-melting-point agarose gels were prepared, respectively, and 1.2% low-melting-point agarose gel was placed on the lower layer of a 6-cm cell culture dish. The 0.6% low-melting-point agarose gel and 5000 K562 cells or RAPSYN-knockout K562 stable expression cells were mixed and placed on the upper layer. After 14 days, the number of colonies formed by the two groups of cells was observed and calculated.

[0062] The results are shown in Figure 6 A-6D.

[0063] Figure 6 As shown in A, CRISPR-Cas9 has completely silenced the RAPSYN gene, and silencing the expression of the RAPSYN gene can significantly inhibit the expression of the BCR-ABL protein. At the same time, Figure 6As shown in B, the in vitro soft agar colony formation experiment showed that after RAPSYN gene silencing, the colony formation ability of the Philadelphia chromosome positive leukemia cells was significantly inhibited.

[0064] Mouse tumor transplantation experiment: wild type K562 cells and RAPSYN knockout K562 stable expression cell lines were used to determine the knockout efficiency, and then 20 five-week-old NCG severely immunodeficient mice were randomly divided into two groups, and each mouse was injected with 1×10 6 cells / 200 μL. After the formation of subcutaneous tumors in mice, the size of the tumor was measured every other day to determine the growth of the tumor. The results showed that the tumor growth rate of the RAPSYN silencing group was significantly reduced compared with the control group. When the tumor volume reached 1500 mm 3 , the tumor tissue was removed, as shown in C, after knocking out the RAPSYN gene, the tumorigenicity of Philadelphia chromosome positive leukemia cells was significantly inhibited. Figure 6

[0065] Mouse survival experiment: wild type K562 cells and RAPSYN knockout K562 stable expression cell lines were used to determine the knockout efficiency, and then 20 five-week-old NCG severely immunodeficient mice were randomly divided into two groups, and each mouse was injected with 1×10 7 cells / 200 μL. The survival status of the mice was observed every day, and the death time of the mice was recorded. The results showed that the survival time of the mice in the RAPSYN silencing group was significantly prolonged compared with the control group. As shown in D, after knocking out the RAPSYN gene, the occurrence and development of Philadelphia chromosome positive leukemia were significantly inhibited. Figure 6

[0066] Example 4: Detection of RAPSYN as E3 ligase mediating neddylation modification of BCR-ABL in three Philadelphia chromosome positive leukemia cells

[0067] The present application first proposes that RAPSYN as E3 ligase can mediate neddylation modification of the corresponding substrate in Philadelphia chromosome positive leukemia cells.

[0068] In three Philadelphia chromosome positive leukemia cells, whether BCR-ABL occurs neddylation modification was detected by immunoprecipitation technology.

[0069] ​​Immunoprecipitation assay: 1 mg of lysed cell protein sample was incubated overnight at 4°C with 10 μg of antibody (BCR-ABL antibody). 40 μL of protein A / Gagrose was transferred to a 1.5 mL centrifuge tube and washed twice with cell lysis buffer. The sample co-incubated with protein lysis buffer and antibody was added to the centrifuge tube and gently incubated at 4°C for 3 hours. After incubation, the sample was washed twice again with cell lysis buffer, and then 60 μL of loading buffer was added to resuspend protein A / Gagrose. The sample was prepared at 100°C for Western blot analysis.

[0070] like Figure 7 As shown in Figure A, NEDD8 can specifically bind to BCR-ABL, inducing its neddylation modification. To demonstrate that RAPSYN acts as an E3 ligase mediating BCR-ABL neddylation modification, changes in BCR-ABL modification levels were detected using RAPSYN knockout cell lines and wild-type cells. Figure 7 As shown in B, the neddylation modification of BCR-ABL was significantly reduced after RAPSYN knockout, indicating that RAPSYN can act as an E3 ligase to mediate the neddylation modification of BCR-ABL in the three Philadelphia chromosome-positive leukemia cell lines.

[0071] Similarly, the neddylation modification of other proteins mediated by RAPSYN as an E3 ligase in leukemia cells is also within the scope of this invention.

[0072] Example 5: Increased RAPSYN expression enhances the stability of BCR-ABL protein.

[0073] To investigate the effect of RAPSYN on the stability of BCR-ABL protein, the following procedures were followed: Wild-type K562 cells and RAPSYN knockout cells were seeded into 35 mm cell culture dishes. When the cell density reached over 60%, actinomycin (100 μg / mL) was added, and the cells were cultured further. Cells were harvested at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h for Western blotting analysis. Figure 8 As shown, the results indicate that the half-life of BCR-ABL protein was significantly reduced after RAPSYN knockout compared with wild-type K562 cells, demonstrating that RAPSYN expression in leukemia cells can increase the stability of BCR-ABL protein.

[0074] Example 6: Targeting the RAPSYN gene to inhibit leukemia stem cells

[0075] To detect the effect of RAPSYN on leukemia stem cells, the following implementation is adopted: wild type K562 cells and RAPSYN knock-out K562 stable expression cell lines are used, after determining the knock-out efficiency, 20 five-week-old NCG severely immunodeficient mice are randomly divided into two groups, and two kinds of cell lines are injected into the tail vein of each mouse, 1×10 7 cells / 200 μL. The state of the mice is observed, and when the mice show poor mental state, the mice are sacrificed, the bone marrow cells are taken, the red blood cells are lysed by red blood cell lysis solution, and are divided into the same type control group and the experimental group, and are respectively blocked for 20 min with FC, then are incubated with the corresponding antibody at 4°C for 20 min, are washed with PBS buffer solution once, and are detected by flow cytometry. Dead cells are excluded, cell adhesion bodies are excluded, the same type control is gated, and Lin - Sca + Kit + population hematopoietic stem cells. As shown in the following table, compared with the control group, knock-out of RAPSYN can significantly reduce the proportion of LSK stem cells in the bone marrow of mice. Figure 9 SEQUENCE LISTING <110> China Pharmaceutical University <120> A new therapeutic target for Philadelphia chromosome positive leukemia and application thereof <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 412 <212> PRT <213> human <400> 1 Met Gly Gln Asp Gln Thr Lys Gln Gln Ile Glu Lys Gly Leu Gln Leu 1 5 10 15 Tyr Gln Ser Asn Gln Thr Glu Lys Ala Leu Gln Val Trp Thr Lys Val 20 25 30 Leu Glu Lys Ser Ser Asp Leu Met Gly Arg Phe Arg Val Leu Gly Cys 35 40 45 Leu Val Thr Ala His Ser Glu Met Gly Arg Tyr Lys Glu Met Leu Lys​ 50 55 60 Phe Ala Val Val Gin He Asp Thr Ala Arg Glu Leu Glu Asp Ala Asp 65 70 75 80 Phe Leu Leu Glu Ser Tyr Leu Asn Leu Ala Arg Ser Asn Glu Lys Leu 85 90 95 Cys Glu Phe His Lys Thr He Ser Tyr Cys Lys Thr Cys Leu Gly Leu 100 105 110 Pro Gly Thr Arg Ala Gly Ala Gin Leu Gly Gly Gin Val Ser Leu Ser 115 120 125 Met Gly Asn Ala Phe Leu Gly Leu Ser Val Phe Gin Lys Ala Leu Glu 130 135 140 Ser Phe Glu Lys Ala Leu Arg Tyr Ala His Asn Asn Asp Asp Ala Met 145 150 155 160 Leu Glu Cys Arg Val Cys Cys Ser Leu Gly Ser Phe Tyr Ala Gin Val 165 170 175 Lys Asp Tyr Glu Lys Ala Leu Phe Phe Pro Cys Lys Ala Ala Glu Leu 180 185 190 Val Asn Asn Tyr Gly Lys Gly Trp Ser Leu Lys Tyr Arg Ala Met Ser 195 200 205 Gln Tyr His Met Ala Val Ala Tyr Arg Leu Leu Gly Arg Leu Gly Ser 210 215 220 Ala Met Glu Cys Cys Glu Glu Ser Met Lys Ile Ala Leu Gin His Gly 225 230 235 240 Asp Arg Pro Leu Gin Ala Leu Cys Leu Leu Cys Phe Ala Asp Ile His 245 250 255 Arg Ser Arg Gly Asp Leu Glu Thr Ala Phe Pro Arg Tyr Asp Ser Ala 260 265 270 Met Ser Ile Met Thr Glu Ile Gly Asn Arg Leu Gly Gin Val Gin Ala 275 280 285 Leu Leu Gly Val Ala Lys Cys Trp Val Ala Arg Lys Ala Leu Asp Lys 290 295 300 Ala Leu Asp Ala Ile Glu Arg Ala Gin Asp Leu Ala Glu Glu Val Gly 305 310 315 320 Asn Lys Leu Ser Gin Leu Lys Leu His Cys Leu Ser Glu Ser Ile Tyr 325 330 335 Arg Ser Lys Gly Leu Gin Arg Glu Leu Arg Ala His Val Val Arg Phe 340 345 350 His Glu Cys Val Glu Glu Thr Glu Leu Tyr Cys Gly Leu Cys Gly Glu 355 360 365 Ser lie Gly Glu Lys Asn Ser Arg Leu Gin Ala Leu Pro Cys Ser His 370 375 380 lie Phe His Leu Arg Cys Leu Gin Asn Asn Gly Thr Arg Ser Cys Pro 385 390 395 400 Asn Cys Arg Arg Ser Ser Met Lys Pro Gly Phe Val 405 410 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <400> 2 gcauugcagg uguggacaaa g 21 <210> 3 <211> 21 <212> RNA <213> Artificial Sequence <400> 3 ggaguguugu gaggagucua u 21 <210> 4 <211> 21 <212> RNA <213> Artificial Sequence <400> 4 ugcacgccag aggcccauuu a 21

Claims

1. The use of synaptic receptor-associated protein RAPSYN as a target in the development or screening of drugs for the treatment of Philadelphia chromosome-positive leukemia, wherein the amino acid sequence of the synaptic receptor-associated protein is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that... The drug is a negative regulator of the RAPSYN protein or its encoding gene.

3. The application as described in claim 2, characterized in that... The negative regulators include protein inhibitors targeting the RAPSYN protein, interfering RNA, gRNA, microRNA, small molecule compound inhibitors, and combinations thereof for nucleic acid aptamers and / or the RAPSYN encoding gene.

4. The application as described in claim 3, characterized in that... The negative regulator is selected from the double strand of the RAPSYN encoding gene. siRNA and / or vector-based shRNA.

5. The application of shRNA encoding the RAPSYN gene in the preparation of drugs for treating Philadelphia chromosome-positive leukemia, characterized in that... The nucleotide sequence of the target sequence that binds to shRNA is shown in any one of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

4.

6. The application as described in claim 5, characterized in that... The drug also contains tyrosine kinase inhibitors.

7. Application of primers for specifically amplifying the synaptic receptor-associated protein RAPSYN gene in the preparation of diagnostic reagents for Philadelphia chromosome-positive leukemia.

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