CN112795651A - MUC20 as marker for diagnosing multiple mantle cell lymphoma proteasome inhibitor drug resistance and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a protein related to proteasome inhibitor drug resistance of multiple mantle cell lymphoma and application of the protein in the proteasome inhibitor drug resistance of the multiple mantle cell lymphoma, wherein the protein is MUC 20. Application of reagent for detecting MUC20 expression level in preparation of proteasome inhibitor drug-resistant product for diagnosing multiple mantle cell lymphoma. According to the invention, the expression of MUC20 gene in PI drug-resistant MCL cell is found to be reduced through GEP analysis for the first time, the direct correlation between MUC20 and PI drug resistance is confirmed through patient data analysis and in vitro tests, and the research in vitro and in animal body confirms that MUC20 participates in PI drug resistance through activating c-Met signal path. Experiments prove that the expression level of MUC20 is directly related to the sensitivity of MCL cells to PI, suggest that MUC20 may be a specific biological index for predicting PI sensitivity and prognosis, and have important theoretical and practical significance for deeply researching the generation mechanism of MUC20 for regulating PI drug resistance.

Description

MUC20 as marker for diagnosing multiple mantle cell lymphoma proteasome inhibitor drug resistance and application thereof

Technical Field

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to a protein related to proteasome inhibitor drug resistance of multiple mantle cell lymphoma and application of the protein in the proteasome inhibitor drug resistance of the multiple mantle cell lymphoma, wherein the protein is MUC 20.

Background

Mantle Cell Lymphoma (MCL) is a hematologic malignancy that includes 3-6% of non-hodgkin's lymphomas. The clinical manifestations of MCL are often aggressive. Over the years, therapeutic strategies have evolved from combination chemotherapy to targeted therapy in combination with immunotherapy. Bortezomib is the FDA-approved first Proteasome Inhibitor (PI) for use in first-line treatment of newly diagnosed and relapsed/refractory MCL. Bortezomib showed effective evidence of response rates as high as 50% in patients with relapsed or refractory MCL compared to previous standard therapy. In an open label phase III study, 487 patients with previously untreated MCL had improved median PFS (24.7 months vs 14.4 months; P P = 0.17) as observed in patients receiving VR-CAP (bortezomib, rituximab, cyclophosphamide, doxorubicin, and prednisone) compared to patients receiving R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Bortezomib as a maintenance therapy may also benefit MCL patients. However, the major and acquired resistance of bortezomib remains a major problem. Therefore, researchers are looking for next generation PIs that can ideally overcome bortezomib resistance.

Carfilzomib is a second generation PI approved by the FDA for the treatment of MM patients. Unlike bortezomib, which binds reversibly, epoxyketones form irreversible binding, which can prolong the duration of proteasome inhibition. Carfilzomib is both resistant and active against relapsed and/or refractory MM, even in some patients receiving bortezomib therapy. Whether carfilzomib is also effective for MCL is under investigation. By high throughput screening, nearly 3800 approved anti-MCL cell line drugs and drug candidates were tested and four drugs (alisertib, carfilzomib, prounostat, and YM155) were identified as having antiproliferative effects on MCL cells in different treatment classes and modes of action. Carfilzomib is a potentially effective drug. Wang et al also demonstrated the in vitro and in vivo therapeutic effects of carfilzomib on MCL. Carfilzomib-induced apoptosis of MCL cells is mediated through activation of JNK, Bcl-2 and mitochondria-associated pathways. In addition, carfilzomib inhibits the growth and survival signaling pathways NF-kB and STAT 3. Zhang et al also demonstrated that carfilzomib-induced apoptosis of MCL cells occurs caspase-dependent via both endogenous and exogenous pathways of caspase. In addition, carfilzomib inhibits constitutive activation of the NF- κ B signaling cascade, whether in MCL cell lines or primary MCL cells, by completely blocking phosphorylation of I κ B α. These data suggest that carfilzomib may be a potentially effective drug for the treatment of MCL.

MUC20 is a member of the mucin (mucin) gene family, and both studies have shown that MUC20 protein expression is associated with IgA nephropathy progression and kidney damage, and increased MUC20 expression is seen during remission periods of ulcerative colitis. Recent researches discover that MUC20 is involved in the occurrence and development process of tumors, and suggest that MUC20 is a potential biological marker of esophageal squamous cell carcinoma, MUC20 expression promotes the development of ovarian epithelial cell carcinoma,MUC20the gene overexpression is related to the recurrence and death of colorectal cancer patients and the like. The C terminal of MUC20 protein is combined with the multifunctional action site of C-Met proto-oncogene, and can inhibit the activation of C-Met signal pathway. But do notMUC20There is no disclosure of a marker for diagnosing resistance to multiple mantle cell lymphoma proteasome inhibitors.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide an application of a protein marker in screening a candidate drug for treating multiple mantle cell lymphoma proteasome inhibitor drug resistance and provide a kit for evaluating the expression level of the protein. In order to investigate the role of proteins associated with resistance to multiple mantle cell lymphoma proteasome inhibitors in resistance to multiple mantle cell lymphoma proteasome inhibitors, appropriate proteins were screened. According to the invention, the expression of MUC20 gene in PI drug-resistant MCL cell is found to be reduced through GEP analysis for the first time, the direct correlation between MUC20 and PI drug resistance is confirmed through patient data analysis and in vitro tests, and the research in vitro and in animal body confirms that MUC20 participates in PI drug resistance through activating c-Met signal path. Experiments prove that the expression level of MUC20 is directly related to the sensitivity of MCL cells to PIs, and suggest that MUC20 may be a specific biological index for predicting PI sensitivity and prognosis, and the important theoretical and practical significance is provided for deeply researching the generation mechanism of MUC20 for regulating PIs drug resistance.

In order to achieve the purpose, the invention adopts the following technical scheme.

Application of reagent for detecting MUC20 expression level in preparation of proteasome inhibitor drug-resistant product for diagnosing multiple mantle cell lymphoma.

Preferably, the reagents are primers that specifically amplify MUC 20.

A kit for diagnosing proteasome inhibitor resistance of multiple mantle cell lymphoma, the kit comprising reagents for detecting the expression level of MUC 20.

Preferably, the reagents are primers that specifically amplify MUC 20.

The use of MUC20 gene, the MUC20 gene is used for preparing the proteasome inhibitor drug-resistant pharmaceutical composition for treating multiple mantle cell lymphoma.

Preferably, the pharmaceutical composition comprises an agonist of the MUC20 gene.

A proteasome inhibitor-resistant pharmaceutical composition for treating multiple mantle cell lymphoma, said pharmaceutical composition comprising an agonist of MUC20 gene.

The agonist of the MUC20 gene is si-MUC20 or sh-MUC 20.

Compared with the prior art, the invention has the following beneficial effects.

The Gene Expression Profiles (GEP) of the invention compare the inhibition of mucin MUC20 identified in the drug-resistant model with drug-free cell lines. MUC20 gene knockdown, resulting in PIs resistance, whereas overexpression thereof is sensitive to carfilzomib or bortezomib. Expression of MUC20 is negatively correlated with activation of c-MET and downstream targets, including p44/42 mitogen-activated protein kinase (MAPK). Activation of c-MET by Hepatocyte Growth Factor (HGF) induces PIs resistance, while inhibition of c-MET restores sensitivity of PIs in vitro and in vivo. Carfilzomib resistance and c-MET activation are associated with increased proteasome activity and with high expression of protease assembly protein, a chaperone protein responsible for catalyzing active proteasome assembly. The link between c-MET and POMP is induced by binding of ETS domain containing protein (ELK) 1 regulated by mapk to POMP promoter. Finally, analysis of the GEP database indicates that high expression of MUC20 correlates with a greater likelihood of achieving and maintaining a bortezomib response. Enhancing proteasome ability by activating c-MET signaling serves as a mechanism for PIs resistance and suggests that expression of MUC20 may be a useful biomarker to distinguish between PIs sensitive and resistant diseases.

The invention firstly verifies that MUC20 can influence the assembly and function of proteasome by regulating POMP expression through detecting the catalytic activity of beta subunit, mature proteasome and POMP expression and other experiments; then experiments such as small molecule inhibitors, activators, key proteins of a knockout signal pathway and the like verify that MUC20 activates a Ras/Raf/MEK/ERK signal pathway through c-Met, and finally activates ELK 1; then ELK1 and ELK are determined by experiments such as reporter gene fluorescent expression, chromatin immunoprecipitation, electrophoretic mobility analysis and the likePOMPThe gene promoter region is combined and promotes the transcription of the gene promoter region to participate in PIs drug resistance; finally, the relationship between MUC20 expression and PIs drug sensitivity and the action mechanism thereof are further verified in a transplanted mouse model.

The invention discovers and confirms for the first timeMUC20The relationship between the gene and the drug resistance of the PIs and the deep exploration of the generation mechanism of the gene have important significance from the improvement of the pharmacological theory of the PIs to the clinical transformation of the PIs to the development of new biological indexes and treatment thinking. Enhancing proteasome ability through activation of c-MET signaling is a mechanism of PIs resistance, and expression of MUC20 may be a useful biomarker to distinguish patients with PI-sensitive and resistant diseases. In addition, the data show that carfilzomib or bortezomib in combination with MET inhibitors may overcome PIs resistance and support their clinical application.

Drawings

FIG. 1 is a comparison of the carfilzomib drug-resistant (CR) MCL cell lines Granta-519/CR and Jeco-1/CR developed according to materials and methods with the same cell lines treated with wild-type (WT) vector. Where a is the exposure of these cells to the indicated concentrations of carfilzomib or vehicle for 24 hours, cell viability was determined using tetrazolium reagent WST-1 to determine mitochondrial activity and thus cell proliferation. All viability data were normalized to vehicle control (0.1% DMSO), arbitrarily set to 1.0, samples were run three times and results are expressed as the mean ± standard error of one of two representative experiments. ", indicates a p value <0.05, CR of all panels was compared to WT cells. B are these same CR cells then exposed to the indicated concentrations of bortezomib or vehicle and analyzed as above.

FIGS. 2A-2D show that NUC20 decreased the expression of MCL cells after PI treatment. A is the content of MUC20 mRNA in the cells detected by quantitative PCR and compared with the WT control without drug. Data points represent Relative Quantitation (RQ) of triplicates ± standard error normalized by GAPDH. The experiment was performed three times and a representative result is shown, wherein ". x" indicates a p value <0.05, and CR of all panels was compared to WT corresponding values. B Western blotting was used to assess the levels of MUC20 protein in WT and CR cell lines, and compared to β -actin as a loading control. C is flow cytometry analysis of expression of CD20+ population MUC20 in bone marrow of MCL patients (newly diagnosed or bortezomib exposed). D is a MUC20 expression analysis of CD20+ cells from 9 patients with MCL. Each symbol represents each patient. Results are expressed as a percentage of MUC20+ cells, and bars represent the average percentage of MUC20 expression. "-" indicates a p value < 0.05.

FIGS. 3A-3D show that MUC20 expression correlates with proteasome inhibitor sensitivity. A is MCL cells infected with either a lentiviral vector without cDNA insertion (OE-con) or cDNA that causes overexpression of MUC20 (OE-MUC 20). Quantitative PCR detection of MUC20 mRNA; b Western blotting to detect MUC20 protein level to determine infection effect. C is the viability of CR and CR/MUC20-OE MCL cells assayed 48 hours after Carfilzomib treatment. D is to randomly divide the immunodeficient mice into two groups, one group was subcutaneously implanted with Granta-519/CR cells, and the other group was subcutaneously implanted with Granta-519/CR/MUC20-OE cells. Both groups were given v CFZ 5 mg/kg, i.p. 2 times per week. The tumor growth was measured with calipers and the tumor volume was calculated with the formula (0.4 '-L' W2).

FIGS. 4A-4D are graphs showing that MET signaling affects the sensitivity of proteasome inhibitors. A is Western blotting to detect the activation states of MET and ERK in Granta-519 and Jeco-1CR or WT cells and CR cell extract over-expressed by muc 20. B is the viability of WT cells assayed 48 hours after exposure to the indicated concentration of carfilzomib in the absence or presence of 100 ng/mL rhHGF, "' indicates a p value <0.05 using WST1 method. E is CFZ with or without ARQ-197 and after 48 hours of irradiation, the viability of Granta-519, Jeco-1 WT and CR cells was examined. F is CFZ with or without ARQ-197(0.1uM) and after 48 hours of irradiation, viability of primary MCL cells was examined.

FIGS. 5A-5C show that MET signaling affects the catalytic activity of proteasomes. A is the detection of chymotrypsin-like (ht-L), caspase-like (C-L) and trypsin-like (T-L) proteasome activity in Granta-519, Jeco-1 WT and CR cells. Data for CR cells were normalized to data for WT cells, arbitrarily set to 1.0, and the results for one of two independent experiments (each experiment was performed in triplicate) are shown, with an "on each panel indicating p < 0.05. B is to detect the proteasome activity of Granta-519 and jeco-1 cells overexpressing MUC20 vector. C for Granta-519 and Jeco-1CR cell treatment, rhHGF 100 ng/mL, or ChT-L, T-L and C-Lproteosomes were examined for proteasomal activity as described above at 1M ARQ 19748 hours. Denotes p value <0.05, compared to control.

FIGS. 6A-6G are ELK1 regulating protease assembly protein expression and carfilzomib resistance. A study on the effect of 100 ng/mL exogenous HGF on the POMP expression level in 36 hours when acting on Granta-519, Jeco-1 WT and CR cells for Western blotting. 1uM ARQ-197 inhibited MET activity in Granta-519, Jeco-1 WT and CR cells for 24 hours and the effect on POMP expression was examined by Western blotting. B is a detailed description of the sequence of the putative binding site for ELK1 disrupted by mutation in the POMP promoter. C is the significance of luciferase reporter gene detection for detecting two ELK1 binding sites in HeLa cells on the activation of the POMP promoter. D was transfected with constructs containing either the empty luciferase promoter (GI3-B) or the POMP promoter containing the entire ELK1 site, the luciferase reporter gene (GI3-POMP), one of the two ELK1 binding site mutations (F-mt and H-mt), or (2 mt). In each experiment, a CMV promoter-renal luciferase reporter was included as a transfection control. "" indicates a p value <0.05 compared to GI 3-POMP. EMSA for E was performed using a 5' biotin-labeled oligonucleotide spanning the transcription start site of the POMP gene, which contained two ELK-1 consensus binding sites (biotin-POMP-p DNA). In the last method, a 5' biotin-labeled oligonucleotide with two ELK-1 binding site mutations (biotin-pomp mutant DNA) was used. Binding reactions were prepared by incubating nuclear extracts of WT or CR cells with the indicated probes (without or with anti-elk1 antibody). F chromatin immunoprecipitation analysis in WT or CR cells using ELK 1-specific antibodies and non-specific immunoglobulin (IgG) as negative controls, or RNA polymerase II (RNA Pol II) antibodies as positive controls. The cells were treated under 100 ng/ml of rhHGF or ARQ-1971 mM for 24 hours. The real-time fluorescent quantitative PCR detects the precipitation of the POMP promoter sequence, and the result is normalized to 10% of input OPM-2/WT. G is the nuclear ELK1 protein level in CR and WT mantle cell lymphoma cells assessed against the histone H3 protein.

Detailed Description

The present invention will be described more fully hereinafter with reference to the following non-limiting examples, which are provided to illustrate and not to limit the present invention, but to which modifications according to the spirit of the invention may be made, which are within the scope of the appended claims.

Examples

Firstly, an experimental method.

1. Development of a carfilzomib-resistant (CR) cell line.

Drug-negative Granta-519 and Jeco-1 mantle cell lymphoma cell lines were initially exposed to carfilzomib at 10% of the Drug's minimal inhibitory concentration (IC 10). After 8 months, the drug concentration was continuously increased from 1nM to 10nM after confirming its proliferative capacity. Once Carfilzomib (CR) Granta-519 and Jeco-1 cell lines were established, all of the above experiments were performed after they were exposed to media without carfilzomib for at least 7 days.

2. Patient sample collection.

The bone marrow is obtained from mantle cell lymphoma patients brought into the bone marrow in Shengjing hospital of Chinese medical university. Informed consent was obtained from all patients and approval by the local ethics committee was obtained according to the declaration of helsinki. And (3) centrifuging and separating the bone marrow mononuclear cells by adopting Ficoll hypha density gradient.

3. Feasibility analysis of cells.

Feasibility analysis of cells. Cell viability assays were as described previously. Carfilzomib, bortezomib, and tivatinib (ARQ-197) were all purchased from seleck chemical company.

4. Real-time RT-PCR.

MUC20 detection probes (batch No. 1107622) were purchased from Applied Biosystems, Inc., and qPCR was performed on Applied Biosystems Step One and real-time PCR systems.

5. Flow cytometry.

Expression of MCL CD20+ cell MUC20 was analyzed by multichannel flow cytometry. Cells were incubated with the monoclonal antibody MUC20 (aa654-684) -PE (LS-C223381). Control mouse IgG for 130 min, and then cells were secondary labeled with PE-conjugated goat anti-mouse IgG for 30 min. Cells were then incubated with APC-conjugated anti-CD 20 mab and fixed in 2% paraformaldehyde. Flow cytometry analysis of stained cells was performed using FACScan and CellQuest Pro software (BD Biosciences).

6. Immunoblotting.

Antibodies include anti-MUC20 from Thermo Fisher Scientific Pierce, anti-phosphor-c-Met and-c-Met from EMD Millipore anti-phosphor-c-Met and-c-Met, anti-phosphor-ERK 1/2 and-ERK 1/2 from Cell Signaling, anti-protein-H3 histone from Santa Cruz Biotechnology, anti-ELK1 from Abcam. Western blot analysis was performed according to standard procedures 30.

7. And (4) measuring the activity of proteasome.

Chymotrypsin-like, trypsin-like and caspase-like activities were detected using proteasome-Glo ­ chymotrypsin-like, trypsin-like and caspase-like cell assay kits (Promega).

8. Lentiviral vector technology and cell transfection.

Using pCDH-CMV-MCS-EF1-CopGFP (System Biosciences; mountain View, CA). The open reading frame of MUC20 was cloned from cDNA (Origene; Rockville, Md.). The lentivirus particle infected cells were classified by flow cytometry. MUUC20 targeting shRNA lentiviral constructs were purchased from Sigma-Aldrich.

9. And (4) detecting luciferase markers.

The POMP promoter was amplified from genomic DNAs POMP-p-Kpn-F, GGGGTACCCTAAGATGTCTCCATCCTGTGG and POMP-p-Bgl2-R, GAAGATCTGTACCCACTCACCATCTTCCGCAGC. KpnI and Bgl2 were then inserted into the pGI 3-based vector. There are two binding sites for ELK1 in the POMP promoter. The pre-mutant (F-mt) construct (CAGGACGGAC GCAC) was obtainedCAGTAA GGGATGTGGG ggccagccccct CGGAAACGGA AGTGAGCGGC), post mutation (H-mt) construct (CAGGACGGAC GCACTTCCGG CGGATGTGGG GGCCAGCCCT) TCAGAATCTG AGTGAGCGGC) and one construct (2mt) in which both mutations occurred. Luciferase activity was monitored by the dual luciferase reporter assay system (Promega) after transfection of constructs into HeLa cells using Lipofectamine 2000 (Life Technologies).

10. Electrophoretic mobility shift analysis.

The 5' biotin-labeled wild-type (WT) probe sequence (WT-f) is: GACGGACGCACTTCCGGCGGATGTGGGGGCCAGCC CTCGGAAACGGAAGTGAGCGGC, spanning the POMP transcription start site, including two ELK1 sites. In the ELK1 mutation site, the sequence (mutf) is: GACGGACGCACCAGTAAGGGATGTGGGGGCCAGCCCTTCAGAATCTGAGTGAGCGGC are provided. protein-DNA binding experiments (Thermo Fisher Scientific) were performed using the LightShift ™ chemiluminescence EMSA kit.

11. Chromatin immunoprecipitation.

Chromatin immunoprecipitation following the manufacturer's protocol (EMD Millipore; Billerica MA). The used Pomp promoter oligonucleotides were F: CGCCGCTCACTTCCGTTT, R: TGGGTCCCTTGGGATTGC are provided. The amplification product spans the POMP transcription initiation site, including the ELK1 binding site.

12. A xenograft model.

Granta-519/CR cells and MUC20 overexpressing Granta-519/CR cells (5X 10)⁶Per) individually, subcutaneously into nude mice (The Jackson Laboratory; Bar Harbor, ME). MiningIntraperitoneal injection is carried out 2 times per week by using carfilzomib (5 mg/kg). Calculating tumor volume = (Width)²X length/2.

13. And (6) counting data.

Statistical analysis was performed using the graphical panel unpaired t-test, with p values <0.05 as significance.

And II, obtaining a result.

1. Production of carfilzomib-resistant cell lymphoma cells.

OPM-2/CR, ANBL-6/CR, KAS-6/1/CR and U266/CR cell lines were generated to explore the possible mechanisms of CR, and this phenotype showed stable drug resistance as shown in FIG. 1A. A similar pattern can be seen when these cells are exposed to bortezomib, as shown in fig. 1B, which shows that this boronic acid proteasome inhibitor decreases activity in CR cells to a lesser extent than WT cells. These findings indicate that some of the resistance mechanisms in these cells are not carfilzomib specific, but are applicable to other of these drugs.

2. MUC20 reduced the expression of MCL cells after PIs treatment.

Differentially expressed genes between CR and WT cells in myeloma cell lines were identified by Gene Expression Profiling (GEP). The GEP data is stored in a GEO library, and the login ID is GSE 62237. Notably, inhibition of MUC20 expression was identified as the most consistent and significant change in all four cell lines. The expression of MUC20 in bortezomib-resistant (BR) ANBL-6/BR, OPM-2/BR, RPMI 8226/BR, and kas 6/1/BR cell lines was compared. A similar pattern also occurs, with significant inhibition of MUC20 associated with the BR phenotype.

The level of MUC20 mRNA in CR cells was determined using qPCR method. western blot results showed a significant reduction in MUC20 transcripts in CR cells, as shown in figure 2A, which correlates with a reduced level of MUC20 protein in CR cells, as shown in figure 2B. Flow cytometry examined the expression of membranous MUC20 in primary specimens of mantle cell lymphoma. Although the expression levels of MUC20 varied between the samples, a reduction in MUC20 expression was found in bortezomib-treated raw samples, as shown in figure 2C. The results show that there are differences in the expression of MUC20 in MCLWT, resistant cell lines and primary samples, and that MUC20 expression was reduced after PIs treatment.

3. MUC20 inhibition is associated with drug resistance.

Then, to determine whether MUC20 plays a direct role in modulating PI sensitivity, MUC20 was overexpressed in drug-resistant cells, as shown in fig. 3A and 3B. Sensitivity to carfilzomib was enhanced when MUC20 was overexpressed in CR cells compared to the empty vector control, as shown in figure 3C. It is also of interest to validate these findings in the xenograft MCL model. Whereas carfilzomib showed stronger activity in the MCL model of MUC20 expressing CR compared to the CRxenogrft model. The reduction in tumor volume was more pronounced in MUC20-OE mice, as shown in FIG. 3D. Taken together, the data support the expression level of MUC20 and the sensitivity of MCL cells to PI.

4. MUC20 inhibits activation of the c-MET signal.

The C-terminus of MUC20 binds to the multifunctional docking site of the MET proto-oncogene and inhibits activation of part of its downstream signaling cascade. Thus, a western blot is used to verify whether this mechanism is also likely to participate in the model of the present invention. Granta-519/CR and Jeco-1/CR cells exhibited higher levels of MET and ERK-1/2 activation, as shown in FIG. 3A. To clarify the effect of MUC20, CR cell extracts overexpressing MUC20 were examined. MUC20 overexpression caused MET and ERK-1/2 to be inactive, as shown in FIG. 3A.

Since HGF exposure mimics some of the downstream effects of the PI resistance phenotype, it was then seen whether HGF could antagonize the therapeutic effect of carfilzomib. Indeed HGF protected both drug-negative Granta-519 and Jeco-1 cells from carfilzomib, which did not reduce cell viability to the same extent in the presence of MET activator, as shown in FIG. 3B. These findings support the hypothesis that c-MET inhibition can restore CR cell sensitivity to carfilzomib. This was studied in a cell line model and it was first found that low concentrations of the C-MET inhibitor ARQ-197 (tivatinib) by itself did not reduce the viability of WT or CR Granta-519 and Jeco-1 cells, as shown in FIG. 3C. However, when cells were exposed to the combined effect of ARQ-197 and carfilzomib, the activity was enhanced. Similar results were also found in the original samples of MCL treated with the c-MET inhibitor ARQ-197 in combination with CFZ, as shown in FIG. 3D. These findings support the possibility that an approach to inhibit c-MET may be an attractive strategy to overcome PI resistance in the clinic.

5. MET signaling affects the catalytic activity of the proteasome.

Carfilzomib binds to the constitutive proteasome beta 5 subunit (PSMB5), which contains chymotrypsin-like activity (ChT-L). Thus, ChT-L activity was first detected in CR cells, and increased ChT-L activity was found in Granta-519/CR and Jeco-1/CR compared to WT controls, as shown in FIG. 5A. In addition, the caspase-like (C-L) and trypsin-like (T-L) activities of these two CR cells were also increased (FIG. 5A). Overexpression of muc20 in CR cells was sufficient by itself to decrease proteasome activity, as shown in FIG. 5B. Further correlating MET signaling with proteasome biology, exposure of Granta-519 and Jeco-1CR cells to HGF increased ChT-L, C-L and T-L activity, as shown in FIG. 5C. Whereas MET signal blockade ARQ-197 decreased ChT-L, C-L and T-L activity in CR cells, as shown in FIG. 5C.

6. Expression of protein combinatorial proteins is associated with MET activity.

These three major proteasome activities are increased in CR cells, suggesting an increased ability of subunits to assemble into functional proteasome particles. The proteinasemblin, also known as POMP, is a key partner responsible for the assembly of the catalytically active beta subunit ring. By Western blotting, protein assembly protein levels in crr cells were increased compared to WT (Granta-519 and Jeco-1), as shown in FIGS. 6A and 6B. Further combining POMP with MET pathway, HGF stimulation increased POMP expression in WT cell lines, and also to some extent in the CR model, as shown in fig. 6A. Whereas the drug MET signal block ARQ-197 decreased POMP levels in WT and CR cells as shown in fig. 6B. Taken together, these data support a link between the c-MET signaling pathway and assembly partners that control the proteasome capacity of myeloma cells.

7. ELK1 regulates the expression of protein assembly protein genes.

To investigate the mechanisms of MET signaling and protein assembly, transcription factor binding sites in the POMP promoter were therefore examined, and a consistent ETS-like gene 1 (ELK1) binding site was found, due to the goal of ELK1 ERK-1/2. Cells were found to be activated at CR, as shown in FIGS. 4A-4D. By Westernblotting, CR cell line nuclear extracts were observed to be enriched in ELK1 protein, as shown in figure 6G. To determine whether ELK1 did affect the expression of POMP, chromatin immunoprecipitation was performed in CR cells using anti-ELK1 antibody, and then sequences near the POMP promoter were detected by PCR. While non-specific iggs did not significantly precipitate these sequences, they contained relatively abundant anti-elk1 antibody, with greater enrichment in CR cells. In addition, HGF increased binding of ELK1 to the POMP promoter, while ARQ-197 decreased binding, as shown in fig. 6D. Biotin-labeled probes and nuclear extracts corresponding to the consensus site of ELK1 were used in gel transfer experiments. A protein-DNA complex was found in WT cells, and when CR cell extracts were used, the complex was present in higher amounts, as shown in FIG. 6C, while an anti-ELK1 antibody hypermigrated the band. Since the proteinamectin promoter has two identical ELK1 sites, as shown in FIG. 6A, a POMP promoter reporter vector with one or two site mutations was prepared and tested for activity in HeLa cells. The wild-type promoter is compared to the empty vector renal blue luciferase reporter gene (GI3-B, shown in FIG. 6B) under basal conditions. Notably, the ELK1 binding site mutation (H-mt, F-mt) at which promoter activity was significantly reduced, and further reduced the level of luciferase activity with the former mutation (2t), the two mutations (F-mt vs 2mt) and H-mt were much less effective. These data indicate that the transcription factor ELK1 binds to the POMP promoter downstream of the c-MET pathway and increases the expression of POMP.

Claims (8)

1. Application of reagent for detecting MUC20 expression level in preparation of proteasome inhibitor drug-resistant product for diagnosing multiple mantle cell lymphoma.

2. The use of claim 1, wherein the reagents are primers for the specific amplification of MUC 20.

3. A kit for diagnosing proteasome inhibitor resistance of multiple mantle cell lymphoma, the kit comprising reagents for detecting the expression level of MUC 20.

4. The kit of claim 3, wherein the reagents are primers that specifically amplify MUC 20.

5. The use of MUC20 gene is characterized in that MUC20 gene is used for preparing a proteasome inhibitor drug-resistant pharmaceutical composition for treating multiple mantle cell lymphoma.

6. The use of the MUC20 gene of claim 6, wherein the pharmaceutical composition comprises an agonist of the MUC20 gene.

7. A proteasome inhibitor resistant pharmaceutical composition for treating multiple mantle cell lymphoma, the pharmaceutical composition comprising an agonist of MUC20 gene.

8. The pharmaceutical composition of claim 7, wherein the agonist of the MUC20 gene is si-MUC20 or sh-MUC 20.

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