Use of POLQ inhibitors in the treatment of multiple myeloma

Abstract

The present invention belongs to the field of biomedicine and specifically discloses the use of POLQ inhibitors in the treatment of multiple myeloma. This disclosure relates to the use of POLQ inhibitors in the preparation of drugs that enhance the sensitivity of multiple myeloma to melphalan treatment, the use of melphalan combined with a POLQ inhibitor in the preparation of drugs for treating multiple myeloma, and drug combinations comprising melphalan and a POLQ inhibitor for treating multiple myeloma. It also relates to the use of preparations for detecting POLQ expression levels in the preparation of products for detecting the resistance of multiple myeloma cells to the cytotoxic effects of melphalan, and the use of highly expressed POLQ in the preparation of biological models for melphalan-resistant multiple myeloma. The POLQ inhibitors disclosed in this disclosure can significantly enhance the therapeutic effect of melphalan, thereby avoiding the excessive dose dependence of efficacy when melphalan is used alone. They can also address the problem of multiple myeloma patients' poor response to melphalan alone, providing new insights into the development of combination therapy strategies for multiple myeloma.

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 proliferation disorder, ranking second in incidence among hematologic malignancies. Advances in treatment over the past two decades have improved the overall prognosis of multiple myeloma, but it remains incurable. Currently, a number of drugs are used clinically to treat multiple myeloma, among which melphalan (CAS: 148-82-3), an alkylating agent that damages DNA, was introduced as a milestone in the treatment of multiple myeloma.

[0003] Melphalan induces DNA interstrand crosslinks (ICLs), hindering the separation of DNA duplexes during replication and transcription, ultimately promoting cell death. However, DNA-damaging agents have poor specificity, and their therapeutic window is often limited by dose-dependent toxic side effects. Furthermore, aberrant activation of the DNA damage response (DDR) machinery 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 is low in activity in normal cells but often overactivated in tumor cells. DNA polymerase θ (POLQ) is a core molecule in the alt-NHEJ repair pathway, using microhomology fragments within the damaged region as primers to mediate DNA gap filling, thereby completing DNA damage repair. Currently, the role of the POLQ-mediated alt-NHEJ pathway in chemotherapy-induced DNA damage repair remains unclear.

[0004] ART558 targets the polymerase domain at the C-terminus of the POLQ protein, while the antibiotic novobiocin (NVB) targets the helicase-like ATPase domain at the N-terminus of the POLQ protein. Therefore, both ART558 and novobiocin are safe and effective POLQ inhibitors. Studies have shown that POLQ inhibition mediated by novobiocin and ART558 can effectively enhance the radiosensitivity of tumor cells, but their role in chemosensitization requires further investigation. Although Chinese patent application CN1923167A previously reported that melphalan, methoxyamine, hydroxylamine, and butylthioninthioxime all had inhibitory effects on breast tumors when used alone at specific concentrations, and also reported that the combination of melphalan with the aforementioned DNA repair enzyme inhibitors (methoxyamine, hydroxylamine, and butylthioninthioxime) enhanced breast tumor inhibition, the effects of these drugs or drug combinations on multiple myeloma remain unclear, and whether methoxyamine, hydroxylamine, and butylthioninthioxime can act as POLQ inhibitors remains unknown.

[0005] The inventors' previous research has found that melphalan can lead to upregulation of POLQ expression in multiple myeloma cells. This phenomenon suggests that the POLQ-mediated alt-NHEJ pathway may be involved in the repair of melphalan-induced DNA damage. It also suggests that activation of this pathway may limit melphalan's killing effect on multiple myeloma, leading to treatment insensitivity or even drug resistance. Given that melphalan is still a widely used multiple myeloma treatment in clinical practice, further research into the causes of poor response and even drug resistance in multiple myeloma treatment is essential, as is the exploration of combination treatment strategies for multiple myeloma. Summary of the Invention

[0006] To address 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] In a first aspect, the present invention provides the use of a POLQ inhibitor in the preparation of a medicament for treating multiple myeloma; wherein the medicament for treating multiple myeloma is a drug for enhancing the sensitivity of multiple myeloma to melphalan treatment. In other words, any use of a POLQ inhibitor to improve the suboptimal sensitivity of multiple myeloma patients to melphalan treatment should be considered within the scope of the present invention. Of course, the use of a POLQ inhibitor as an adjuvant to melphalan treatment for multiple myeloma in profitable treatment should also be considered as using 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 novobiocin, ART558, ART812, ART4215, and RP-6685.

[0011] Secondly, the effect of the POLQ inhibitor on the sensitivity of melphalan therapy 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 tumor cells are less sensitive to the drug and thus affect the efficacy of the drug when melphalan is used alone (a manifestation of drug resistance); the use of POLQ inhibitors as therapeutic drugs does not require excessive reliance on increasing the therapeutic dosage of melphalan, and can produce significant therapeutic effects at low doses.

[0012] Special note: The noun "at least" means that it can be any one or more of the listed options, 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 in combination with a POLQ inhibitor in the preparation of a medicament for treating multiple myeloma, any combination of melphalan and a POLQ inhibitor is within the scope of the present invention, and the form of the new drug is not limited. The new drug may be a combination or a set of two independently packaged drugs with a combined use indication (e.g., a combined use indication for treating multiple myeloma, or the combination appears in the same prescription). Some of the features may be specifically selected or limited as follows:

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

[0016] Secondly, the POLQ inhibitor acts to enhance the sensitivity of multiple myeloma cells to melphalan. Specifically, 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 and achieving the desired effect at a lower dosage.

[0017] Third, the concentration ratio of melphalan to the POLQ inhibitor is generally 2.5-20:50-150. In some specific examples, the concentration ratios of melphalan to the POLQ inhibitor novobiocin for the L-363 cell line are 2.5-20 μM:75-150 μM, and for the RPMI-8226 cell line are 2.5-20 μM:50-125 μM. Furthermore, for both L-363 and RPMI-8226 cell lines, the concentration ratios of melphalan to the POLQ inhibitor ART558 are 2.5-20 μM:2.5-20 μM. The effective concentration of melphalan can be 2.5-20 μM, which can be adjusted based on clinical needs and is not strictly limited.

[0018] Regarding a drug combination for treating multiple myeloma, it comprises the active ingredients melphalan and a POLQ inhibitor. The POLQ inhibitor is at least one of novobiocin, ART558, ART812, ART4215, and RP-6685. The concentration ratio of melphalan to the POLQ inhibitor in this drug combination for treating multiple myeloma can be referenced to the above description; the effective concentration of melphalan can be 2.5 to 20 μM. As before, the form of the drug combination is not particularly required; any combination drug is considered within the scope of this invention.

[0019] Regarding the use of drug combinations in the preparation of products for in vitro killing of multiple myeloma cells for non-diagnostic and therapeutic purposes, the drug combinations contain the active ingredients melphalan and a POLQ inhibitor. However, the resulting products are generally not used as pharmaceuticals, but primarily as in vitro preparations, such as in vitro experimental preparations, and are not intended for diagnosis or treatment of diseases. Such products are more commonly used in commercial experiments, where the combination of melphalan and a POLQ inhibitor is used to kill multiple myeloma cells in vitro to adjust experimental variables. These drug combinations also exhibit a more pronounced killing effect than melphalan alone. Specifically, the POLQ inhibitor is at least one of novobiocin, ART558, ART812, ART4215, and RP-6685. 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, including a mixture formed in a culture dish during use, which should be considered a use of the present invention. The multiple myeloma cells are at least one of L-363 cells and RPMI-8226 cells.

[0020] A third aspect of the present invention provides the use of a preparation for detecting POLQ expression levels in the preparation of a product for detecting the resistance of multiple myeloma cells to melphalan. A high POLQ expression level indicates strong resistance to melphalan, while a low POLQ expression level indicates weak resistance. The method detects whether POLQ expression levels are upregulated or downregulated. Upregulation indicates strong resistance to melphalan, while downregulation indicates weak resistance. In practical applications, the POLQ expression level in multiple myeloma cells without melphalan can be used as a reference to assess whether expression levels are upregulated or downregulated. Furthermore, the term "preparation" as used in this section is not limited to traditional preparation concepts; any product that can achieve the intended purpose of testing falls within the scope of this invention.

[0021] A fourth aspect of the present invention provides the use of POLQ in preparing a biological model of multiple myeloma melphalan-resistant cells. High expression of POLQ reduces the sensitivity of multiple myeloma cells to melphalan therapy. This allows the development of a biological model demonstrating resistance to melphalan-induced cell death in multiple myeloma cells, providing a model for other studies, such as investigating whether certain substances can ameliorate this resistance to melphalan therapy, thereby developing strategies to improve melphalan-resistant and insensitive multiple myeloma cells.

[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 commonly used cell models for in vitro multiple myeloma research and can be used to further develop a melphalan treatment resistance model.

[0025] In the present disclosure, POLQ inhibitors can significantly enhance the therapeutic effect of melphalan, thereby avoiding the problem of excessive dependence of the efficacy 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 new ideas 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 shPOLQ cell lines were transfected with lentiviral vectors (A, B), the CCK-8 assay was used to detect the proliferation inhibitory effect of melphalan on L-363shPOLQ and control cell lines (C) and 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 viability of L-363 cell lines (A) and RPMI-8226 cell lines (B).

[0029] Figure 4 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 ART558 on cell viability in L-363 cell lines (A) and RPMI-8226 cell lines (B).

[0030] Figure 5 Figure 2 shows the synergistic effect scores of the CCK-8 cell proliferation assay results of L-363 cell lines (A) and RPMI-8226 cell lines (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 Figure 2 shows the synergistic effect scores of the CCK-8 cell proliferation assay 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 ART558. A Bliss score greater than 0 is considered to have a synergistic effect.

[0032] Figure 7 Figure 3 is the result 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 Annexin V FITC / PI double staining.

[0033] Figure 8 Figure 3 is the result of flow cytometry detection of the effects of melphalan and POLQ inhibitor ART558 on cell apoptosis in L-363 cell line (A) and RPMI-8226 cell line (B) when treated with them alone or in combination using Annexin V FITC / PI double staining.

[0034] Figure 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] Figure 10 Figure 3 is the result of alkaline comet assay detecting 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] Figure 11 Figure 1 shows the anatomical appearance of the tumor (A) and the tumor growth curve (B) in a 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 will be described in detail below with reference to specific examples.

[0038] Improving the efficacy of melphalan is a pressing challenge in the treatment of multiple myeloma. Designing drug combinations that combine DNA-damaging agents with DDR inhibitors is expected to provide new insights into the development of combination therapy strategies for multiple myeloma. In this study, Western blotting revealed that melphalan induces activation of the POLQ-mediated alt-NHEJ pathway in multiple myeloma cells. Subsequently, a lentiviral vector was used to construct a shPOLQ cell line. Cell proliferation assays demonstrated that knockdown of POLQ significantly increased the sensitivity of multiple myeloma cells to melphalan. Cell proliferation, synergy analysis, and apoptosis assays further demonstrated the in vitro synergistic anti-multiple myeloma effect of melphalan in combination with the POLQ inhibitors novobiocin or ART558. Alkaline comet assays demonstrated that the combination of melphalan and a POLQ inhibitor induced more severe DNA damage than either agent alone. Furthermore, the in vivo synergistic anti-multiple myeloma effect of the melphalan-novobiocin combination was validated in a subcutaneous mouse 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 assay 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 / 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 6-well culture plates to ensure a final DMSO concentration of 1‰ in each well. The plates were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0042] (3) Cells were collected, total cell protein was extracted, and Western Blotting experiments were performed to detect the effect of melphalan treatment on the expression level of POLQ (POLQ antibody Abmart, TD13563), a key molecule of the alt-NHEJ pathway, 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 in the results, 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 the 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 POLQ knockdown L-363 or RPMI-8226 stable cell lines

[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 knocks down an irrelevant gene and the expression of POLQ is not affected, but the background expression level of POLQ in L-363 and RPMI-8226 cell lines is higher than that in normal human cells;

[0047] (3) After 12 hours of transfection, fresh culture medium was replaced and cultured for 48 hours, and then puromycin was added for stable strain selection;

[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 / mL;

[0050] (6) The final concentrations of melphalan were set to 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM. The drug was mixed with L-363shControl (RPMI-8226shControl) and L-363shPOLQ (RPMI-8226shPOLQ) cell suspensions to ensure a final DMSO concentration of 1‰. The cells were evenly inoculated into 96-well plates at a volume of 100 μl / 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 a 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 strains were successfully constructed; Figure 2 As shown in Figures C and D, melphalan significantly enhanced the growth inhibitory effect of multiple myeloma cells with low POLQ expression compared with the control group, indicating that the L-363shPOLQ and RPMI-8226shPOLQ cell lines were significantly more sensitive to melphalan than the control group; and as the melphalan treatment concentration increased, the difference in the tumor cell killing effect between the low-expression group and the control group became more obvious, indicating 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, allowing melphalan to achieve a more ideal killing effect at lower concentrations, thereby reducing the dependence on melphalan concentration during the treatment of multiple myeloma.

[0053] Example 3: CCK-8 assay was used to detect the effects of different concentrations of melphalan alone or different concentrations of melphalan combined with specific concentrations of the POLQ inhibitor novobiocin / ART558 on cell viability 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 / 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 concentrations of melphalan were set at 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, respectively;

[0060] Melphalan combined with the POLQ inhibitor ART558:

[0061] ① Melphalan (MEL) single-drug group: the final concentrations of melphalan were set at 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 the 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 the 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) The drug and cell suspension were mixed to ensure a final DMSO concentration of 1‰, and 100 μl / well was evenly inoculated into a 96-well plate, with three replicate wells set for each drug concentration;

[0064] (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 the CCK8 kit.

[0065] Experimental results: Figure 3As shown in , 2.5-10 μM melphalan alone has a very limited inhibitory effect on the growth of L-363 cells, while when melphalan is combined with the POLQ inhibitor novobiocin (NVB), it has a significant effect on the growth inhibition of L-363 cells. Melphalan alone has a certain growth inhibitory effect on RPMI-8226 cells, and when 5-20 μM melphalan is combined with novobiocin (NVB), the growth inhibition effect of RPMI-8226 cells is significantly enhanced. 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, but it had a certain growth inhibitory effect on RPMI-8226 cells. However, 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, the POLQ inhibitors novobiocin (NVB) and ART558 significantly enhanced 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 POLQ activity 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 lines 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 / 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 concentrations of melphalan were set at 0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM;

[0073] ③ Novobiocin (NVB) single-drug group: For the L-363 cell line, the final concentration of NVB was set to 0 μM, 75 μM, 100 μM, 125 μM, and 150 μM; for the 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-mentioned different concentrations of MEL and NVB were arranged and combined;

[0075] Melphalan combined with the POLQ inhibitor ART558:

[0076] ①Control group: add DMSO;

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

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

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

[0080] (3) The drug and cell suspension were mixed to ensure a final DMSO concentration of 1‰, and 100 μl / well was evenly inoculated into a 96-well plate, with three 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), cell viability was measured using the CCK8 assay. Combefit software was used to analyze drug synergy. A Bliss score > 0 indicated a synergistic effect between the two drugs at that concentration.

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

[0083] The Bliss scores of the drug combination analysis of melphalan and ART558 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 analysis of the effects of melphalan and POLQ inhibitor novobiocin / ART558 alone or in combination on the apoptosis rate of L-363 / RPMI-8226 cell lines

[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 / mL;

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

[0088] Melphalan combined with the POLQ inhibitor novobiocin:

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

[0090] Melphalan combined with the 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 (concentrations 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 and cell suspension were mixed to ensure a final DMSO concentration of 1‰, and the cells were evenly inoculated into 12-well plates at a volume of 1 mL / well. The cells were 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, and stained with Annexin V-FITC / PI (BD, USA) kit. The cell apoptosis level was detected by a microscope and the cell apoptosis rate was calculated.

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

[0095] Example 6: Alkaline comet assay to detect the effects of melphalan and the 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 / 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 (concentrations of 10 μM + 100 μM);

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

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

[0101] (5) Prepare the gel layers using the Comet Assay Kit (ELK Biotechnology) according to the following table:

[0102] First layer of gel 100 μL 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 dish, 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, pour in freshly prepared alkaline electrophoresis buffer, and leave at room temperature for 60 minutes to allow the DNA to unwind under alkaline conditions and produce alkaline-denaturable segments, making it easier for DNA chain breaks 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 slides in a dish. Immerse the slides in 0.4 mM Tris-HCl buffer and neutralize them three times at 4°C for 5 minutes each time. After neutralization, add 10 μL of PI staining solution to each slide, cover with a coverslip, and stain in the dark for 10 minutes.

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

[0109] Experimental results: Figure 10 As shown in the figure, the comet tail moment of the melphalan and novobiocin combined treatment group was significantly higher than that of the single drug treatment group, indicating that the combined treatment of melphalan and novobiocin can induce more severe 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 armpit of NSG mice to establish 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 monotherapy group (MEL), a novobiocin monotherapy group (NVB), and a combination drug group for a total of two weeks. On the 1st, 2nd, 4th, and 5th day of each week, the mice in the novobiocin monotherapy group and the combination drug 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 monotherapy group and the combination drug group were intraperitoneally injected with 2 mg / kg melphalan, and the rest of the mice were intraperitoneally injected with normal saline; and rest was allowed 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 counted;

[0116] Experimental results: Figure 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 improved compared with other single-drug groups, especially the tumor volume of the melphalan and novobiocin combination group was the 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, upregulation of the POLQ-mediated alt-NHEJ repair pathway in multiple myeloma cells may help cells resist the killing effects of the DNA-damaging agent melphalan. Knockdown of POLQ expression or blocking the alt-NHEJ pathway with a POLQ inhibitor significantly enhances the anti-multiple myeloma effect of melphalan. The combination of melphalan and a POLQ inhibitor exhibits strong synergistic effects, significantly inhibiting cell proliferation and promoting apoptosis by inducing the accumulation of DNA damage.

[0118] It will be apparent 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. Such modifications fall within the scope of protection of the present invention. The protection scheme of the present invention shall be subject to the claims appended hereto.

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 that enhances the sensitivity of multiple myeloma to melphalan treatment; the POLQ inhibitor is at least one of novobiocin, ART558, ART812, ART4215, and RP-6685.

2. Use of melphalan combined with a POLQ inhibitor in the preparation of a drug for treating multiple myeloma; wherein, The POLQ inhibitor is at least one of novobiocin, ART558, ART812, ART4215, and RP-6685.

3. The use according to claim 2; wherein The effect of the POLQ inhibitors is shown to enhance the sensitivity of multiple myeloma cells to melphalan treatment.

4. 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 novobiocin, ART558, ART812, ART4215, and RP-6685.

5. 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; the POLQ inhibitor is at least one of novobiocin, ART558, ART812, ART4215, and RP-6685.

6. The use according to claim 5, characterized in that The multiple myeloma cells are at least one of L-363 cells and RPMI-8226 cells.

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