Use of POLQ inhibitors in treatment of multiple myeloma

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses application of a POLQ inhibitor in treatment of multiple myeloma. The invention relates to an application of a POLQ inhibitor in preparation of a medicine for improving the Melphalan treatment sensitivity of multiple myeloma, an application of Melphalan combined with the POLQ inhibitor in preparation of a medicine for treating multiple myeloma, and a medicine composition containing Melphalan and the POLQ inhibitor and used for treating multiple myeloma. The invention also relates to an application of a preparation for detecting the expression level of POLQ in preparation of a product for detecting the killing effect of multiple myeloma cells for resisting melphalan and an application of high-expression POLQ in preparation of a biological model for resisting treatment of multiple myeloma melphalan. The POLQ inhibitor can significantly enhance the therapeutic effect of melphalan, so that the problem that the curative effect excessively depends on dosage when melphalan is independently applied is avoided, the problem that multiple myeloma is poor in response to single melphalan can also be solved, and a new thought is provided for development of a multiple myeloma combined treatment strategy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of a POLQ inhibitor in treating multiple myeloma. Background Art

[0002] Multiple myeloma is a clonal plasma cell abnormal proliferation disease, and its incidence ranks second among hematological malignancies. In the past two decades, with the advancement of treatment methods, the overall prognosis of multiple myeloma has improved, but it is still incurable. Currently, there are many drugs used in the clinic to treat multiple myeloma, among which melphalan (CAS: 148-82-3) is an alkylating agent that damages DNA. Its introduction is a milestone in the history of multiple myeloma treatment.

[0003] Melphalan induces DNA interstrand crosslinks (ICLs) damage, hinders the separation of DNA double strands during replication and transcription, and ultimately promotes cell death. However, DNA damaging agents have poor specificity, and their therapeutic window is often limited by dose-dependent toxic side effects. In addition, the abnormal activation of the DNA damage response (DDR) mechanism in tumor cells promotes their adaptability to DNA damaging agents and is one of the main causes of treatment resistance. Alternative non-homologous end joining (alt-NHEJ) repair is a low-fidelity, error-prone DNA damage repair pathway that has very low activity in normal cells and is often overactivated in tumor cells. DNA polymerase θ (POLQ) is a core molecule in the alt-NHEJ repair pathway, which uses micro-homologous fragments in the damaged region as primers to mediate the filling of DNA gaps and then complete the repair of DNA damage. At present, the role of the POLQ-mediated alt-NHEJ pathway in the repair of chemotherapy-induced DNA damage is still unclear.

[0004] ART558 targets the polymerase domain at the C-terminus of the POLQ protein, and the antibiotic novobiocin (NVB) can target the helicase-like ATPase domain at the N-terminus of the POLQ protein. Therefore, both ART558 and novobiocin can be used as a safe and effective POLQ inhibitor. At present, studies have shown that POLQ inhibition mediated by novobiocin and ART558 can effectively enhance the radiosensitivity of tumor cells, but its role in chemotherapy sensitization needs further study. Although it was previously reported in Chinese patent application CN1923167A that melphalan and methoxyamine, hydroxylamine, and butylthionine thioxime all had inhibitory effects on breast tumors when used alone at specific concentrations, it was also reported that when melphalan was used in combination with the above-mentioned DNA repair enzyme inhibitors (methoxyamine, hydroxylamine, and butylthionine thioxime), the inhibitory effect on breast tumors could be enhanced, but the effects of the above-mentioned drugs or drug combinations on multiple myeloma are still unclear, and it is still unknown whether methoxyamine, hydroxylamine, and butylthionine thioxime can be used as POLQ inhibitors.

[0005] The inventors' previous studies have found that melphalan can lead to upregulation of POLQ expression in multiple myeloma cells. This phenomenon indicates that the alt-NHEJ pathway mediated by POLQ may be involved in the repair of melphalan-induced DNA damage, and also indicates that the activation of this pathway may limit the killing effect of melphalan on multiple myeloma, leading to treatment insensitivity or even drug resistance. Given that melphalan is still a widely used drug for the treatment of multiple myeloma in clinical practice, it is necessary to further study the reasons for its poor response or even treatment resistance during the treatment of multiple myeloma, and explore combined treatment strategies for multiple myeloma. Summary of the invention

[0006] In view of the above problems, the present invention provides the use of POLQ inhibitors in the treatment of multiple myeloma, mainly to solve the problems of large dose-dependent toxic side effects, poor efficacy, and drug resistance in the current clinical treatment of multiple myeloma with melphalan.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides the use of a POLQ inhibitor in the preparation of a drug for treating multiple myeloma; wherein the drug for treating multiple myeloma is a drug for improving the sensitivity of multiple myeloma to melphalan treatment. In other words, any use of a POLQ inhibitor to improve the poor sensitivity of multiple myeloma to melphalan treatment should be within the scope of the present invention. Of course, if a POLQ inhibitor is used as an adjuvant in the treatment of multiple myeloma with melphalan in a profitable treatment behavior, it should also be regarded as the use of this technical solution.

[0009] The following selections or limitations can be made for some of the features:

[0010] In one of them, the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685.

[0011] Secondly, the effect of the POLQ inhibitor on the therapeutic sensitivity of melphalan can be manifested as follows: by inhibiting the activity of POLQ, the resistance of multiple myeloma cells to the killing effect of melphalan is reduced, thereby making the therapeutic efficacy of melphalan better, avoiding the phenomenon that when melphalan is used alone, the tumor cells are prone to poor drug sensitivity and thus affect the drug efficacy (manifestation of drug resistance); the use of POLQ inhibitors as therapeutic drugs does not need to rely too much on increasing the therapeutic dosage of melphalan, and can produce obvious therapeutic effects at low doses.

[0012] Special note: The noun "at least" means that it can be any one or more of the options listed, or it can be equivalent options other than those listed (the same below).

[0013] The second aspect of the present invention provides the use of melphalan combined with a POLQ inhibitor in the preparation of a drug for treating multiple myeloma and a pharmaceutical composition for treating multiple myeloma.

[0014] Regarding the use of melphalan combined with a POLQ inhibitor in the preparation of a drug for treating multiple myeloma, any use of melphalan combined with a POLQ inhibitor in a drug for treating multiple myeloma is within the scope of the present invention, and the form of the new drug is not limited. It can be a combination or a drug set with two independent packages but with a combined use indication (for example, there is a prompt for combined use to treat multiple myeloma, or it appears in the same prescription). Some of the features can be specifically selected or limited as follows:

[0015] In one of them, the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685.

[0016] Second, the effect of the POLQ inhibitor is to enhance the therapeutic sensitivity of multiple myeloma cells to melphalan, specifically by inhibiting the activity of POLQ to reduce the resistance of multiple myeloma cells to the killing effect of melphalan, thereby making the therapeutic efficacy of melphalan better and achieving the desired effect with a lower dosage.

[0017] Among them, the concentration ratio of melphalan and POLQ inhibitor is generally 2.5-20:50-150. In some specific examples, the concentration ratio of melphalan and POLQ inhibitor neomycin for L-363 cell line is 2.5-20μM:75-150μM, and for RPMI-8226 cell line is 2.5-20μM:50-125μM; In addition, for L-363 and RPMI-8226 cell lines, the concentration ratio of melphalan and POLQ inhibitor ART558 is 2.5-20μM:2.5-20μM. As for melphalan, its effective concentration can be 2.5-20μM, which can be adjusted according to clinical needs without strict limitation.

[0018] As for the drug combination for treating multiple myeloma, it contains the active ingredients melphalan and a POLQ inhibitor. Among them, the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685. In the drug combination for treating multiple myeloma, the concentration ratio of melphalan and POLQ inhibitor can refer to the content recorded in the previous paragraph; the effective concentration of melphalan can be 2.5 to 20 μM. As before, there is no special requirement for the form of the drug combination, and all those that actually form a combination drug form should be within the scope of the present invention.

[0019] As for the application of the drug combination in the preparation of a product for killing multiple myeloma cells in vitro for non-diagnostic and therapeutic purposes, the drug combination contains the active ingredients melphalan and a POLQ inhibitor, but the formed product is generally not used as a drug, but mainly as an in vitro preparation, such as an in vitro experimental preparation, and its purpose is not to diagnose and treat the disease. This type of product is more used in commercial experiments. In the experiment, the composition formed by the combination of melphalan and a POLQ inhibitor is used to kill multiple myeloma cells in vitro to achieve the purpose of adjusting the experimental variables, and the drug combination has a more obvious killing effect than melphalan alone. Specifically: the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, RP-6685, etc. The composition referred to in this article is not necessarily limited to a mixture in the traditional sense. It can be a powder or liquid mixture, and it also includes the two forming a mixture in a culture dish when used, which should actually be regarded as the use of the scheme of the present invention. The multiple myeloma cells are at least one of L-363 cells and RPMI-8226 cells.

[0020] The third aspect of the present invention provides the use of a preparation for detecting the expression level of POLQ in the preparation of a product for detecting the resistance of multiple myeloma cells to the killing effect of melphalan; wherein, if the expression level of POLQ is high, the multiple myeloma cells have a strong resistance to the killing effect of melphalan, or if the expression level of POLQ is low, the multiple myeloma cells have a weak resistance to the killing effect of melphalan. By detecting whether the expression level of POLQ is upregulated or downregulated, if it is upregulated, it indicates that the multiple myeloma cells have a strong resistance to the killing effect of melphalan, and if it is downregulated, it indicates that the multiple myeloma cells have a weak resistance to the killing effect of melphalan. In practical applications, the expression level of POLQ in multiple myeloma cells when melphalan is not used can be used as a reference to evaluate the upregulation or downregulation of the expression level. In addition, the "preparation" mentioned in this section is not limited to the traditional concept of preparations, and all products that can achieve the purpose of detection should be within the scope of the present invention.

[0021] The fourth aspect of the present invention provides the use of POLQ in preparing a biological model of resistance to melphalan treatment of multiple myeloma; wherein the high expression of POLQ reduces the sensitivity of multiple myeloma cells to melphalan treatment. Based on this, a biological model with the phenomenon of multiple myeloma cells resisting melphalan killing can be formed, and then a research model can be provided for other studies, such as studying whether there are certain substances that can improve this phenomenon of resistance to melphalan killing, so as to develop strategies that can improve the insensitivity and drug resistance of multiple myeloma to melphalan treatment.

[0022] The following selections or limitations can be made for some of the features:

[0023] In one of them, the biological model is at least a multiple myeloma cell model.

[0024] In another embodiment, the cell model is at least a multiple myeloma L-363 cell model or a multiple myeloma RPMI-8226 cell model. Both the multiple myeloma L-363 cell model and the multiple myeloma RPMI-8226 cell model are common cell models used for in vitro research of multiple myeloma, and a melphalan treatment resistance model can be further formed based on them.

[0025] In the present disclosure, the POLQ inhibitor can significantly enhance the therapeutic effect of melphalan, thereby avoiding the problem of excessive dependence of the therapeutic effect of melphalan on concentration when treated alone, and can also solve the problem of poor response of multiple myeloma to melphalan alone. The pharmaceutical composition provides a new idea for the development of combined treatment strategies for multiple myeloma; at the same time, it also provides a detection and analysis method for the drug resistance phenomenon of melphalan in the treatment of multiple myeloma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Western Blotting experiments were performed to detect the effects of different concentrations of melphalan on the expression of POLQ, a key molecule in the alt-NHEJ pathway, in L-363 cell lines (A) and RPMI-8226 cell lines (B).

[0027] Figure 2 After the shPOLQ cell line was constructed by transfection of lentiviral vector (A, B), the CCK-8 method was used to detect the proliferation inhibitory effect of melphalan on L-363shPOLQ and control cell lines (C), RPMI-8226shPOLQ and control cell lines (D).

[0028] Figure 3 The CCK-8 assay was used to detect the effects of different concentrations of melphalan alone or different concentrations of melphalan combined with a specific concentration of the POLQ inhibitor novobiocin on the cell survival rate of L-363 cell line (A) and RPMI-8226 cell line (B).

[0029] Figure 4 The CCK-8 method was used to detect the effects of different concentrations of melphalan alone or different concentrations of melphalan combined with a specific concentration of the POLQ inhibitor ART558 on cell survival in L-363 cell lines (A) and RPMI-8226 cell lines (B).

[0030] Figure 5 The synergistic effect score diagram of the CCK-8 cell proliferation experiment results of L-363 cell line (A) and RPMI-8226 cell line (B) treated with different concentrations of melphalan and different concentrations of the POLQ inhibitor novobiocin. A Bliss score greater than 0 is considered to have a synergistic effect.

[0031] Figure 6 The synergistic effect score diagram of the CCK-8 cell proliferation experiment results of L-363 cell line (A) and RPMI-8226 cell line (B) treated with different concentrations of melphalan and different concentrations of POLQ inhibitor ART558. A Bliss score greater than 0 is considered to have a synergistic effect.

[0032] Figure 7 The results of flow cytometry detection of the effects of melphalan and the POLQ inhibitor novobiocin on cell apoptosis in L-363 cell line (A) and RPMI-8226 cell line (B) when treated with them alone or in combination using AnnexinV FITC / PI double staining.

[0033] Figure 8 The graphs show the results of flow cytometry detection of the effects of melphalan and POLQ inhibitor ART558 alone or in combination on apoptosis in L-363 cell line (A) and RPMI-8226 cell line (B) using Annexin V FITC / PI double staining.

[0034] Fig. 9 Statistical graphs of apoptosis results of L-363 cell lines (A, C) and RPMI-8226 cell lines (B, D) treated with melphalan and POLQ inhibitor novobiocin / ART558 alone or in combination.

[0035] Fig.10 The results of alkaline comet assay for DNA damage induced by melphalan and POLQ inhibitor novobiocin alone or in combination in L-363 cell line (A) and RPMI-8226 cell line (B).

[0036] Fig.11 Figure 1 shows the anatomical appearance of the tumor (A) and the tumor growth curve (B) in the mouse multiple myeloma subcutaneous tumor model after treatment with melphalan and the POLQ inhibitor novobiocin alone or in combination. DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to specific examples.

[0038] Improving the efficacy of melphalan is a difficult problem to be solved in the treatment of multiple myeloma. Designing a drug combination of DNA damaging agents and DDR inhibitors is expected to provide new ideas for the development of combined treatment strategies for multiple myeloma. In this study, Western Blotting experiments were used to find that melphalan can induce the activation of the POLQ-mediated alt-NHEJ pathway after acting on multiple myeloma cells. Subsequently, shPOLQ cell lines were constructed using lentiviral vectors. Cell proliferation experiments showed that knocking down POLQ can significantly increase the sensitivity of multiple myeloma cells to melphalan. Cell proliferation, synergy analysis and cell apoptosis experiments were further used to prove the in vitro synergistic anti-multiple myeloma effect of the drug combination of melphalan and POLQ inhibitors neomycin or ART558; the alkaline comet experiment proved that the drug combination of melphalan and POLQ inhibitors can induce more severe DNA damage than single drugs; the in vivo synergistic anti-multiple myeloma effect of the drug combination of melphalan and neomycin was further verified by a mouse subcutaneous tumor model. Melphalan in the examples of this part was purchased from Dalian Meilun Biotechnology Co., Ltd., novobiocin was purchased from Selleck, and ART558 was purchased from MCE.

[0039] Example 1: Western Blotting experiment to detect the effect of different concentrations of melphalan on the expression of POLQ, a key molecule in the alt-NHEJ pathway, in L-363 or RPMI-8226 cell lines

[0040] (1) L-363 cells (purchased from DSMZ Cell Bank, Germany) or RPMI-8226 cells (purchased from ATCC Cell Bank, USA) in the logarithmic growth phase were collected and the cell density was adjusted to 1×10 5 Pieces / mL;

[0041] (2) Melphalan was dissolved in DMSO, and the final concentrations of melphalan were set to 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. After mixing with 2 mL of cell suspension, the cells were inoculated into a 6-well culture plate to ensure that the final concentration of DMSO in each well was 1‰. The plates were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0042] (3) Collect cells, extract total cell protein, and perform Western Blotting experiments to detect the effect of melphalan treatment on the expression level of POLQ (POLQ antibody Abmart, TD13563), a key molecule in the alt-NHEJ pathway, in order to clarify the activation of the alt-NHEJ pathway in L-363 cells or RPMI-8226 cells after melphalan treatment.

[0043] Experimental results: Figure 1 As shown, the expression level of POLQ in L-363 cells or RPMI-8226 cells was significantly increased after melphalan treatment, and the expression level of POLQ also increased with the increase of melphalan treatment concentration, indicating that melphalan-induced DNA damage promoted the activation of POLQ-mediated alt-NHEJ pathway in multiple myeloma cells, and the degree of activation increased with the increase of melphalan concentration, which in turn led to an increase in the degree of resistance to melphalan treatment.

[0044] Example 2: CCK-8 assay to detect the inhibitory effect of melphalan on the proliferation of L-363 or RPMI-8226 cells with POLQ knockdown

[0045] (1) Collect L-363 cells or RPMI-8226 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 / mL, 1mL of cell suspension was inoculated into a 12-well culture plate;

[0046] (2) The control group lentivirus shControl and the interference group lentivirus shPOLQ were transfected into the well plates respectively, where: shControl knocked down an irrelevant gene and the expression of POLQ was not affected, but the background expression level of POLQ in L-363 and RPMI-8226 cell lines was higher than that in normal human cells;

[0047] (3) 12 h after transfection, fresh medium was replaced and cultured for 48 h, and then puromycin was added for stable strain screening;

[0048] (4) After 10 days of continuous screening, cells were collected, total cell protein was extracted, and Western Blotting experiments were performed to detect the expression of POLQ in L-363 and RPMI-8226 cells to determine the interference effect;

[0049] (5) Collect L-363 cells or RPMI-8226 cells stably transfected with shControl and shPOLQ and adjust the cell density to 1×10 5 Pieces / mL;

[0050] (6) The final concentrations of melphalan were set to 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. The drugs were mixed with L-363shControl (RPMI-8226shControl) and L-363shPOLQ (RPMI-8226shPOLQ) cell suspensions to ensure that the final concentration of DMSO was 1‰. The cells were evenly inoculated in a 96-well plate at a volume of 100 ul / well, and three replicate wells were set for each drug concentration.

[0051] (7) After culturing in a 37°C, 5% CO2 incubator for 24 h (L-363 cells) or 48 h (RPMI-8226 cells), the cell viability was detected using the CCK8 kit (MCE).

[0052] Experimental results: Figure 2 As shown in A and B, the POLQ expression levels in L-363shPOLQ and RPMI-8226shPOLQ cell lines were significantly lower than those in L-363shControl and RPMI-8226shControl cell lines, indicating that the interference strain was successfully constructed; Figure 2 As shown in C and D, the inhibitory effect of melphalan on the growth of multiple myeloma cells with low POLQ expression was significantly enhanced compared with the control group, indicating that the sensitivity of L-363shPOLQ and RPMI-8226shPOLQ cell lines to melphalan was significantly enhanced compared with the control group; and with the increase of melphalan treatment concentration, the difference in the killing effect on tumor cells between the low expression group and the control group became more obvious, which also showed that inhibiting POLQ expression can more significantly weaken the resistance of multiple myeloma cells to the killing effect of melphalan when the melphalan concentration increases, so that melphalan can achieve a more ideal killing effect at a lower concentration, thereby reducing the dependence on melphalan concentration during the treatment of multiple myeloma.

[0053] Example 3: CCK-8 method was used to detect the effect of different concentrations of melphalan alone or different concentrations of melphalan combined with specific concentrations of POLQ inhibitor novobiocin / ART558 on cell survival rate in L-363 / RPMI-8226 cell lines

[0054] Experimental steps:

[0055] (1) Collect L-363 cells or RPMI-8226 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 Pieces / mL;

[0056] (2) The specific groups are as follows:

[0057] Melphalan combined with the POLQ inhibitor novobiocin:

[0058] ① Melphalan (MEL) single-drug group: the final concentration of melphalan was set to 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM;

[0059] ② Melphalan (MEL) + novobiocin (NVB) group, NVB was dissolved in DMSO, and the final concentrations of MEL + NVB were set to 0 μM + 100 μM, 2.5 μM + 100 μM, 5 μM + 100 μM, 10 μM + 100 μM and 20 μM + 100 μM;

[0060] Melphalan combined with POLQ inhibitor ART558:

[0061] ① Melphalan (MEL) single-drug group: the final concentration of melphalan was set to 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM;

[0062] ② Melphalan (MEL) + ART558 (ART) group, ART was dissolved in DMSO. For L-363 cell line, the final concentrations of MEL + ART were set to 0 μM + 20 μM, 2.5 μM + 20 μM, 5 μM + 20 μM, 10 μM + 20 μM and 20 μM + 20 μM, respectively; for RPMI-8226 cell line, the final concentrations of MEL + ART were set to 0 μM + 5 μM, 2.5 μM + 5 μM, 5 μM + 5 μM, 10 μM + 5 μM and 20 μM + 5 μM, respectively;

[0063] (3) Mix the drug and cell suspension to ensure that the final concentration of DMSO is 1‰, and evenly inoculate them in a 96-well plate at a volume of 100 ul / well, with 3 replicate wells set for each drug concentration;

[0064] (4) After culturing in a 37° C., 5% CO 2 incubator for 24 h (L-363 cells) or 48 h (RPMI-8226 cells), the cell viability was detected using the CCK8 kit.

[0065] Experimental results: Figure 3As shown in the results, 2.5-10 μM melphalan alone had very limited inhibitory effect on the growth of L-363 cells, while melphalan combined with the POLQ inhibitor novobiocin (NVB) had a significant effect on the growth inhibition of L-363 cells. Melphalan alone had a certain growth inhibition effect on RPMI-8226 cells, and 5-20 μM melphalan combined with novobiocin (NVB) significantly enhanced the growth inhibition effect on RPMI-8226 cells. Figure 4 As shown in the results, the growth inhibitory effect of 2.5-10 μM melphalan alone on L-363 cells did not change significantly, and it had a certain growth inhibitory effect on RPMI-8226 cells. When melphalan was used in combination with ART558, it had a significant effect on the growth inhibitory effect of both L-363 cells and RPMI-8226 cells. Figure 3 , Figure 4 As shown in the data, compared with melphalan monotherapy, POLQ inhibitors novobiocin (NVB) and ART558 can significantly enhance the growth inhibitory effect of melphalan (MEL) on L-363 cells and RPMI-8226 cells, indicating that when POLQ expression is upregulated, the sensitivity of multiple myeloma to melphalan treatment is relatively weak (i.e., multiple myeloma cells are more resistant to the killing effect of melphalan), and inhibiting the activity of POLQ can enhance the sensitivity of multiple myeloma to melphalan treatment (i.e., reduce the resistance of multiple myeloma cells to the killing effect of melphalan).

[0066] Example 4: Synergistic effect analysis of CCK-8 cell proliferation assay results of L-363 / RPMI-8226 cell line treated with different concentrations of melphalan and different concentrations of POLQ inhibitor novobiocin / ART558

[0067] Experimental steps:

[0068] (1) Collect L-363 cells or RPMI-8226 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 Pieces / mL;

[0069] (2) The specific groups are as follows:

[0070] Melphalan combined with the POLQ inhibitor novobiocin:

[0071] ①Control group: add DMSO;

[0072] ②Melphalan (MEL) single-drug group: the final concentration of melphalan was set to 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM;

[0073] ③ Novobiocin (NVB) single-drug group: for L-363 cell line, the final concentration of NVB was set to 0 μM, 75 μM, 100 μM, 125 μM and 150 μM; for RPMI-8226 cell line, the final concentration of NVB was set to 0 μM, 50 μM, 75 μM, 100 μM and 125 μM;

[0074] ④ Melphalan (MEL) + novobiocin (NVB) group, the above different concentrations of MEL and NVB were arranged and combined;

[0075] Melphalan combined with POLQ inhibitor ART558:

[0076] ①Control group: add DMSO;

[0077] ②Melphalan (MEL) single-drug group: the final concentration of melphalan was set to 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM;

[0078] ③ART558 (ART) monotherapy group: the final concentration of ART was set to 0 μM, 2.5 μM, 5 μM, 10 μM and 20 μM;

[0079] ④ Melphalan (MEL) + ART558 (ART) group, the above different concentrations of MEL and ART were arranged and combined;

[0080] (3) Mix the drug and cell suspension to ensure that the final concentration of DMSO is 1‰, and evenly inoculate them in a 96-well plate at a volume of 100 ul / well, with 3 replicate wells set for each drug concentration;

[0081] (4) After culturing in a 37°C, 5% CO2 incubator for 24 h (L-363 cells) or 48 h (RPMI-8226 cells), the cell viability was detected using a CCK8 kit. Combenefit software was used to analyze the drug combination effect. A Bliss score > 0 means that the two drugs have a synergistic effect at this drug concentration.

[0082] Experimental results: Figure 5 and Figure 6 As shown, melphalan and novobiocin were present at various concentrations.

[0083] The Bliss scores of the drug combination analysis of melphalan and POLQ inhibitors were all >0, indicating that the combination of melphalan and POLQ inhibitors has a significant synergistic killing effect on multiple myeloma cells.

[0084] Example 5: Annexin V FITC / PI double staining flow cytometry detection of the effect of melphalan and POLQ inhibitor novobiocin / ART558 alone or in combination on the apoptosis rate of L-363 / RPMI-8226 cell line

[0085] Experimental steps:

[0086] (1) Collect L-363 cells and RPMI-8226 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 Pieces / mL;

[0087] (2) The specific groups are as follows

[0088] Melphalan combined with the POLQ inhibitor novobiocin:

[0089] A control group (DMSO), a melphalan (MEL) single-drug treatment group (concentration of 10 μM), a novobiocin (NVB) single-drug treatment group (concentration of 100 μM), and a melphalan (MEL) + novobiocin (NVB) combined treatment group (concentration of 10 μM + 100 μM) were set up;

[0090] Melphalan combined with POLQ inhibitor ART558:

[0091] A control group (DMSO), a melphalan (MEL) single-drug treatment group (concentration of 10 μM), an ART558 (ART) single-drug treatment group (concentration of 20 μM for L-363 cell line and 5 μM for RPMI-8226 cell line), and a melphalan (MEL) + ART558 (ART) combined treatment group (concentration of 10 μM + 20 μM for L-363 cell line and 10 μM + 5 μM for RPMI-8226 cell line) were set up;

[0092] (3) The drug was mixed with the cell suspension to ensure that the final concentration of DMSO was 1‰, and the mixture was evenly inoculated into a 12-well plate at a volume of 1 mL / well and cultured in a 37°C, 5% CO2 incubator for 24 h (L-363 cells) or 48 h (RPMI-8226 cells);

[0093] (4) After the treatment, the cells were collected by centrifugation, washed once with PBS, stained with Annexin V-FITC / PI (BD, USA) kit, and the cell apoptosis level was detected by computer and the cell apoptosis rate was calculated.

[0094] Experimental results: Figure 7 to Figure 9 As shown, the combination of melphalan and POLQ inhibitor neomycin / ART558 induced a more obvious cell apoptosis effect compared with the single-drug treatment group, and POLQ inhibitor can significantly enhance the pro-apoptotic effect of melphalan on multiple myeloma cells.

[0095] Example 6: Alkaline comet assay to detect the effect of melphalan and POLQ inhibitor novobiocin alone or in combination on the level of intracellular DNA damage in L-363 / RPMI-8226 cell lines

[0096] Experimental steps:

[0097] (1) Collect L-363 cells and RPMI-8226 cells in the logarithmic growth phase and adjust the cell density to 1×10 5 Pieces / mL;

[0098] (2) Set up a control group (DMSO), a melphalan (MEL) single-drug treatment group (concentration of 10 μM), a novobiocin (NVB) single-drug treatment group (concentration of 100 μM), and a melphalan (MEL) + novobiocin (NVB) combined treatment group (concentration of 10 μM + 100 μM);

[0099] (3) The drug was mixed with the cell suspension to ensure that the final concentration of DMSO was 1‰, and the mixture was evenly inoculated into a 6-well plate at a volume of 2 mL / well and cultured in a 37°C, 5% CO2 incubator for 24 h (L-363 cells) or 48 h (RPMI-8226 cells);

[0100] (4) After treatment, cells were collected by centrifugation, washed once with PBS, and resuspended in PBS to adjust the cell density to 1x10 6 Pieces / mL;

[0101] (5) Prepare the gel layers according to the table below using the comet assay kit (ELK Biotechnology):

[0102] First layer of gel 100 μL of normal melting point agarose (NMA) Second layer of gel 10 μL cells and 75 μL low melting point agarose (LMA) The third layer of gel 75 μL low melting point agarose LMA

[0103] Add three layers of gel on the slide in this way, cover with a cover glass after each addition of gel, and place at 4℃ to solidify;

[0104] (6) Cell lysis: Remove the coverslip, place the slide in a plate, pour in pre-cooled Lysis Buffer, lyse at 4°C for 1 h, remove the slide and rinse with PBS;

[0105] (8) DNA alkaline unwinding: Place the slide in a horizontal electrophoresis tank and pour in freshly prepared alkaline electrophoresis buffer. Leave at room temperature for 60 min to allow the DNA to unwind under alkaline conditions and produce alkaline-denaturable segments, making it easier for the DNA broken chains to migrate in the electric field.

[0106] (8) Single cell electrophoresis: voltage 25 V, electrophoresis 30 min;

[0107] (9) Neutralization and staining: After electrophoresis, place the slide in a dish. Immerse the slide in 0.4 mM Tris-HCl buffer and neutralize three times at 4°C for 5 min each time. After neutralization, add 10 μL of PI staining solution to each slide, cover with a coverslip, and stain for 10 min in the dark.

[0108] (10) Observation, photography, and analysis: A fluorescence microscope was used with an excitation light wavelength of 515 to 560 nm. 25 cells were randomly selected from each sample and the comet tail moment was measured.

[0109] Experimental results: Fig.10 As shown in the figure, the comet tail moment of the group treated with melphalan and neomycin was significantly higher than that of the group treated with single drugs, indicating that the combined treatment of melphalan and neomycin can induce more serious DNA damage in multiple myeloma cells, thereby promoting cell death.

[0110] Example 7: Construction of a mouse subcutaneous tumor model to detect the in vivo anti-multiple myeloma effects of melphalan and the POLQ inhibitor novobiocin alone or in combination.

[0111] Experimental steps:

[0112] (1) Take 8×10 6 RPMI-8226 cells were inoculated subcutaneously in the right axilla of NSG mice to construct a subcutaneous tumor model;

[0113] (2) After the tumors were measurable, the mice were randomly divided into a normal saline group (Vehicle, normal saline), a melphalan group (MEL), a novobiocin group (NVB), and a combination group for a total of two weeks of treatment. On the 1st, 2nd, 4th, and 5th day of each week, the mice in the novobiocin group and the combination group were intraperitoneally injected with 100 mg / kg novobiocin, and the rest of the mice were intraperitoneally injected with normal saline; on the 3rd and 6th day of each week, the mice in the melphalan group and the combination group were intraperitoneally injected with 2 mg / kg melphalan, and the rest of the mice were intraperitoneally injected with normal saline; and the mice rested on the 7th day;

[0114] (3) Measure the size of the subcutaneous tumor of the mouse every other day and calculate the tumor volume V (mm 3 )=1 / 2×major axis (mm)×minor axis (mm)^2;

[0115] (4) After the treatment, the mice were killed, the tumors were removed, and the results were statistically analyzed;

[0116] Experimental results: Fig.11As shown in the mouse tumor anatomy diagram (A) and tumor growth curve diagram (B), the effect of the combination of melphalan and novobiocin was significantly optimized compared with other single-drug groups, especially the tumor volume of the melphalan and novobiocin combination group was most significantly reduced compared with the control group, indicating that the combination of melphalan and novobiocin also showed good synergistic anti-multiple myeloma effect in the in vivo model.

[0117] In summary, the upregulation of the alt-NHEJ repair pathway mediated by POLQ in multiple myeloma cells may help cells resist the killing effect of the DNA damaging agent melphalan. Knocking down the expression of POLQ or using POLQ inhibitors to block the alt-NHEJ pathway can significantly enhance the anti-multiple myeloma effect of melphalan. The drug combination of melphalan and POLQ inhibitors showed a strong synergistic effect, significantly inhibiting cell proliferation and promoting cell apoptosis by inducing the accumulation of DNA damage.

[0118] It will be clear to those skilled in the art that various modifications to the above embodiments may be made without departing from the overall spirit and concept of the present invention. All of these modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims attached to the present invention.

Claims

1. Use of a POLQ inhibitor in the preparation of a drug for treating multiple myeloma; wherein: The drug for treating multiple myeloma is a drug for improving the sensitivity of multiple myeloma to melphalan treatment.

2. The use according to claim 1; wherein The POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685.

3. Use of melphalan combined with POLQ inhibitor in the preparation of drugs for the treatment of multiple myeloma.

4. The use according to claim 3; wherein: The POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685.

5. The use according to claim 3; wherein: The effect of the POLQ inhibitor is manifested in enhancing the sensitivity of multiple myeloma cells to melphalan treatment.

6. A drug combination for treating multiple myeloma, characterized in that: The invention comprises the active ingredients melphalan and a POLQ inhibitor; wherein the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685.

7. Use of a drug combination for non-diagnostic and therapeutic purposes in the preparation of a product for killing multiple myeloma cells in vitro; characterized in that: The drug combination comprises the active ingredients melphalan and a POLQ inhibitor.

8. The use according to claim 7; the POLQ inhibitor is at least one of neomycin, ART558, ART812, ART4215, and RP-6685; the multiple myeloma cells are at least one of L-363 cells and RPMI-8226 cells.

9. Use of a preparation for detecting the expression level of POLQ in the preparation of a product for detecting the resistance of multiple myeloma cells to the killing effect of melphalan; wherein: If the POLQ expression level is high, the multiple myeloma cells are more resistant to the killing effect of melphalan, or if the POLQ expression level is low, the multiple myeloma cells are less resistant to the killing effect of melphalan.

10. Application of highly expressed POLQ in the preparation of a biological model of multiple myeloma melphalan-resistant therapy; wherein, The high expression of POLQ can reduce the therapeutic sensitivity of multiple myeloma cells to melphalan; the biological model is at least a multiple myeloma cell model; the cell model is at least a multiple myeloma L-363 cell model or a multiple myeloma RPMI-8226 cell model.

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