Use of ifi16 gene as a target in preparation of drug for treating bortezomib-resistant multiple myeloma

Abstract

This invention provides the application of the IFI16 gene as a target in the preparation of bortezomib-resistant drugs for the treatment of multiple myeloma, involving drug targets, related diagnostic reagents, and treatment strategies for the diagnosis and treatment of multiple myeloma (MM). This invention reveals for the first time the core driving role of IFI16 in bortezomib resistance in MM and its novel mechanism of action through the Wnt / β-catenin pathway; more importantly, it provides novel biomarkers, drug targets, and highly feasible combination therapy regimens for accurate prognostic assessment of MM and overcoming the clinically challenging problem of bortezomib resistance, possessing significant theoretical implications and broad clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug target, related diagnostic reagents, and treatment strategies for the diagnosis and treatment of multiple myeloma (MM), particularly the application of the interferon-gamma-induced protein 16 (IFI16) gene and its mediated Wnt / β-catenin signaling pathway in predicting and overcoming resistance to bortezomib (BTZ) in multiple myeloma. Background Technology

[0002] Multiple myeloma is the second most common hematologic malignancy, and bortezomib, a proteasome inhibitor, is its first-line treatment. However, the vast majority of patients eventually develop bortezomib resistance, leading to treatment failure and disease relapse. Currently, the specific molecular mechanisms of bortezomib resistance are not fully understood, and effective predictive biomarkers and methods to overcome resistance are lacking in clinical practice. Therefore, identifying the key molecules driving bortezomib resistance and developing corresponding intervention strategies is of significant clinical importance.

[0003] IFI16 is a protein involved in the regulation of innate immunity and epigenetics. Its pro-cancer or anti-cancer effects in solid tumors have been reported. However, the functional role of IFI16 in multiple myeloma has not been systematically studied, and its regulatory role and related molecular mechanisms in bortezomib resistance remain unclear.

[0004] Therefore, it is necessary to further explore the mechanism of action of the IFI16 gene and its expression in multiple myeloma, and to further propose solutions for bortezomib resistance in multiple myeloma. Summary of the Invention

[0005] In light of this, this invention reveals for the first time the core driving role of IFI16 in bortezomib resistance in multiple myeloma (MM) and its novel mechanism of action through the Wnt / β-catenin pathway. It also provides a novel biomarker, drug target, and highly feasible combination therapy for accurate prognostic assessment of MM and overcoming the clinically challenging problem of bortezomib resistance.

[0006] One of the objectives of this invention is to provide the IFI16 gene or its expression product as a biomarker for the preparation of products for the diagnosis or prognostic assessment of multiple myeloma.

[0007] Furthermore, the product is used to detect the expression level of IFI16 in a sample.

[0008] Specifically, the samples include bone marrow fluid, peripheral blood, and isolated myeloma cells.

[0009] The second objective of this invention is to provide the IFI16 gene or its expression product as a biomarker for screening or preparing products for the treatment of multiple myeloma.

[0010] Furthermore, the product can overcome or reverse bortezomib resistance.

[0011] A third objective of this invention is to provide a pharmaceutical composition comprising active ingredient A and active ingredient B. The active ingredient A is an IFI16 inhibitor and / or a Wnt / β-catenin signaling pathway inhibitor; The active ingredient B is bortezomib and / or other pharmaceutically acceptable proteasome inhibitors.

[0012] Furthermore, the IFI16 inhibitor includes small molecule compounds, small interfering RNA, short hairpin RNA, antisense oligonucleotides, and specific antibodies targeting IFI16.

[0013] Furthermore, the Wnt / β-catenin signaling pathway inhibitor is IWP-2 or a pharmaceutically acceptable derivative thereof.

[0014] Furthermore, the drug also includes a pharmaceutically acceptable carrier; The carrier includes phospholipids, cholesterol, PEG lipids for preparing siRNA lipid nanoparticles, physiological saline and glucose solution for injection, surfactants, and cyclodextrin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) A novel key molecule driving bortezomib resistance and its novel mechanism of action were discovered.

[0016] Novel Target Molecule: Previous studies have largely focused on known oncogenes, drug-metabolizing enzymes, or classic apoptosis / autophagy pathways to explore MM drug resistance. This invention is the first to discover and demonstrate that the immune-related gene IFI16 is a key gene upregulated in MM cells in response to bortezomib stress and directly mediates acquired drug resistance, expanding the molecular spectrum of MM drug resistance research.

[0017] Novel Mechanism of Action: Previous studies have primarily linked the role of IFI16 in solid tumors to the STING pathway, viral infection, or cellular senescence. This invention reveals a novel functional mechanism of IFI16 in hematologic malignancies for the first time: it promotes cell survival and proliferation by activating the classic Wnt / β-catenin signaling pathway, thereby mediating drug resistance. This discovery of the "IFI16-Wnt / β-catenin" axis provides a completely new signaling pathway perspective for understanding multivariate (MM) resistance.

[0018] (2) It provides a new and effective biomarker for predicting the efficacy and prognosis of bortezomib.

[0019] This invention, through clinical sample cohort analysis, clarifies that high expression of IFI16 is significantly associated with adverse clinical characteristics (such as advanced ISS stage, high tumor burden, bone disease, and renal impairment) and shorter survival in MM patients, and is an independent prognostic factor in multivariate analysis.

[0020] Compared to currently used but more macroscopic indicators in clinical practice (such as ISS staging and cytogenetic abnormalities), detecting IFI16 expression levels provides a more direct and molecular tool for predicting an individual patient's initial response to bortezomib and the risk of drug resistance, which helps to achieve more precise prognostic stratification and treatment decisions.

[0021] (3) A novel treatment strategy and drug combination for overcoming bortezomib resistance were proposed.

[0022] A novel targeted therapy strategy: This invention not only identifies IFI16 as a target, but also demonstrates through functional experiments that directly knocking down or inhibiting IFI16 can effectively reverse drug resistance and restore the sensitivity of MM cells to bortezomib. This provides direct theoretical basis and experimental support for the development of novel targeted therapies against IFI16 (such as small molecule inhibitors and gene silencing therapies).

[0023] Novel Combination Therapy: This invention elucidates that the downstream Wnt / β-catenin pathway is a key effector in IFI16-mediated drug resistance. Therefore, a strategy of combining existing Wnt pathway inhibitors (such as IWP-2) with bortezomib is proposed, and this strategy has been validated in in vitro models to effectively overcome drug resistance caused by IFI16 overexpression. This provides a potentially transformative therapeutic combination for addressing bortezomib resistance in clinical practice.

[0024] (4) It has achieved a complete logical closed loop from basic discovery to clinical validation, and its translational medical value is clear.

[0025] The research path of this invention is complete: starting from clinical problems (drug resistance), candidate molecules (IFI16) are discovered through omics screening, systematic clinical relevance verification is carried out, in-depth functional and mechanistic studies are conducted, and finally, the effectiveness of the treatment strategy is verified in animal models.

[0026] This research model makes the findings of this invention not only scientifically innovative but also possess clear prospects for clinical translation. IFI16 can be used as a prognostic biomarker for patient stratification and as a therapeutic target for new drug development. Combination therapy of its downstream pathway inhibitors with existing standard treatments is even more realistically feasible. Attached Figure Description

[0027] Figure 1 This is a graph showing the abnormally high expression of IFI16 in Example 1 of the present invention and its clinical correlation with MM, wherein: A represents the expression level of IFI16 mRNA in bone marrow samples from newly diagnosed multiple myeloma (NDMM), complete remission (CR), relapse (R) patients, and iron deficiency anemia (IDA) controls, detected by qRT-PCR. B represents the qRT-PCR analysis of IFI16 mRNA levels in bone marrow samples from the same MM patient at different disease stages. Correlation analysis of CD IFI16 expression level and clinical indicators of NDMM patients; E represents Kaplan-Meier analysis of progression-free survival (PFS) based on IFI16 expression levels; F represents the Kaplan-Meier survival analysis curves of three independent MM patient cohorts stratified based on IFI16 expression levels.

[0028] Figure 2 This is a diagram showing the results of Western blotting (WB) detection of bortezomib-induced IFI16 expression in MM cells in Example 1 of this invention, wherein: A represents the protein expression level of IFI16 in H929 cells after treatment with different concentrations of bortezomib, as detected by Western blot. B represents the protein expression level of IFI16 in U266 cells after treatment with different concentrations of bortezomib, as detected by Western blot.

[0029] Figure 3 This is a graph showing the immunohistochemical detection results of IFI16 expression in newly diagnosed and relapsed MM in Example 1 of the present invention, wherein: A shows the immunohistochemical (IHC) staining results of IFI16 in bone marrow samples from NDMM and IDA control groups; B shows the immunohistochemical (IHC) staining results of IFI16 in paired samples from different disease stages of the same patient.

[0030] Figure 4 The results of PCR and WB detection and screening of IFI16 interfering RNA sequences in Example 2 of this invention are as follows: A represents the efficiency of IFI16 knockdown in H929 and U266 cells after siRNA transfection, verified by qRT-PCR. B is a Western blot (WB) assay to verify the knockdown efficiency of IFI16 in H929 and U266 cells after siRNA transfection.

[0031] Figure 5 The results of PCR and WB detection of IFI16 overexpression efficiency in Example 2 of this invention are as follows: A is to verify the transfection efficiency of IFI16 in H929 and U266 cells after viral transfection by qRT-PCR; B is a Western blot (WB) assay to verify the transfection efficiency of IFI16 in H929 and U266 cells after viral transfection.

[0032] Figure 6 In Example 2 of this invention, the CCK8 assay was used to detect the effect of specifically knocking down FI16 gene expression on MM cell proliferation and BTZ IC50. 50 The impact, including: A represents the proliferation capacity of MM cells after IFI16 knockdown at different time points, which was detected by CCK-8 assay. B represents cell proliferation at different time points after IFI16 overexpression, as detected by CCK-8 assay. C represents bortezomib IC50 in cells from the control group and the IFI16 knockdown group. 50 Value analysis; D represents the empty control group and bortezomib IC50 in IFI16 overexpressing cells. 50 Value analysis.

[0033] Figure 7 In Example 2 of this invention, flow cytometry was used to detect the effect of inhibiting / overexpressing IFI16 on MM cell apoptosis, wherein: A represents the analysis of apoptosis in si-NC and IFI16 knockdown of H929 and U266 cells with or without bortezomib treatment using Annexin V / PI flow cytometry. B represents the apoptosis of vector and IFI16-overexpressing H929 and U266 cells with and without bortezomib treatment, analyzed by Annexin V / PI flow cytometry.

[0034] Figure 8 In Example 2 of this invention, flow cytometry was used to detect the effect of inhibiting / overexpressing IFI16 on the MM cell cycle, wherein: A represents the cell cycle distribution of si-NC and IFI16 knockdown H929 and U266 cells with or without bortezomib treatment, analyzed by flow cytometry. B represents the cell cycle distribution of vector and IFI16-overexpressing H929 and U266 cells with and without bortezomib treatment, analyzed by flow cytometry.

[0035] Figure 9 In Example 3 of this invention, the luciferase reporter gene assay was used to detect the effect of inhibiting / overexpressing FI16 gene expression on the WNT pathway, wherein: A represents the detection of Wnt / β-catenin signaling activity in IFI16 knocked-down H929 and U266 cells under BZT treatment or no treatment conditions using a TOPflash / FOPflash luciferase reporter assay. B represents the detection of Wnt / β-catenin signaling activity in IFI16-overexpressing H929 and U266 cells under BZT treatment or no treatment conditions using a TOPflash / FOPflash luciferase reporter assay.

[0036] Figure 10 In Example 3 of this invention, a Western blot (WB) experiment was conducted to detect the effect of inhibiting / overexpressing FI16 gene expression on WNT pathway molecular expression, wherein: A represents the expression levels of Wnt / β-catenin signaling pathway-related proteins (Wnt1, GSK-3β, p-GSK-3β, and β-catenin) in IFI16 knockdown cells under BTZ treatment or no treatment conditions, detected by Western blot. B represents the expression levels of Wnt / β-catenin signaling pathway-related proteins (Wnt1, GSK-3β, p-GSK-3β, and β-catenin) in IFI16-overexpressing cells under BTZ treatment or no treatment conditions, detected by Western blot.

[0037] Figure 11 In Example 3 of this invention, the WNT pathway inhibitor can partially restore the effects of IFI16 gene overexpression on WNT pathway molecules and cell proliferation, wherein: A represents the Western blot analysis of Wnt pathway-related protein expression in IFI16-overexpressing cells treated with IWP-2 and / or bortezomib. BC represents the proliferative capacity of IFI16-overexpressing MM cells after IWP-2 treatment (B) and further combined with bortezomib treatment (C) as assessed by CCK-8 assay.

[0038] Figure 12 In Example 3 of this invention, the WNT pathway inhibitor can partially restore the effects of overexpressed IFI16 gene on WNT pathway molecules, apoptosis, and cell cycle, wherein: A and B represent the apoptosis (A) and cell cycle distribution (B) of IFI16-overexpressing MM cells after combined treatment with IWP-2 and bortezomib, analyzed by flow cytometry.

[0039] Figure 13 In Example 4 of this invention, an animal MM model experiment was conducted to study the effect of targeting the IFI16 gene and the combination of BTZ on MM, wherein: A is a schematic diagram of a systemic dissemination model: luciferase-labeled U266 cells (vector or IFI16-OE) are injected into NCG mice via tail vein, and bortezomib (0.5 mg / kg) or PBS is administered every 4 days; A is a schematic diagram of a subcutaneous xenograft model: H929 cells (si-NC or siIFI16-S3) are inoculated into nude mice, and bortezomib or PBS is administered. B represents monitoring tumor burden using bioluminescence imaging; C represents the recording of mouse weight changes; DF represents the image of the removed tumor (D), quantitative analysis of tumor volume (E), and tumor growth curve (F). G represents the immunohistochemical (IHC) staining analysis of subcutaneous xenograft tumor tissues under different treatment conditions for IFI16, Ki67, β-catenin, and Cyclin D1. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0041] Example 1 This embodiment provides the detection and application of the IFI16 gene and its expression product as a biomarker for bortezomib resistance and prognosis. The specific experiment is as follows: 1.1 Patient Sample Collection Bone marrow samples were collected from patients with multiple myeloma (MM) (including newly diagnosed, remission, and relapsed / refractory stages) and controls with iron deficiency anemia (IDA). Total RNA was extracted and cDNA was obtained by reverse transcription. The expression level of IFI16 mRNA was detected by real-time quantitative PCR (qRT-PCR) using specific primers.

[0042] The primer sequence for IFI16 is: Positive 5'-CCCGAGAAACAATGACCCCA-3' (SEQ ID NO:1) Reverse 5'-TTGGTGTTGTAGGCAAGTGG-3' (SEQ ID NO:2); The primer sequence for the internal reference GAPDH is as follows: Positive 5'-AGCAAGAGCACAAGAGGAAG-3' (SEQ ID NO:3). Reverse 5'-GGTTGAGCACAGGGTACTTT-3' (SEQ ID NO:4).

[0043] qPCR reaction system:

[0044] qPCR reaction procedure:

[0045] The relative expression level was calculated using the 2^(-ΔΔCt) method.

[0046] Results: IFI16 expression was significantly higher in newly diagnosed and relapsed / refractory MM patients than in IDA control and remission patients. Figure 1 AB); expression levels were positively correlated with ISS stage, β2-microglobulin, bone disease, renal impairment, and other adverse clinical features. Figure 1 CD); Kaplan-Meier survival analysis showed that patients with high IFI16 expression had shorter progression-free survival (PFS) and overall survival (OS). Figure 1 EF).

[0047] 1.2 Cell line validation Human MM cell lines (U266 and H929) in logarithmic growth phase were treated with different concentrations (0, 2.5, 5, 10 nM) of bortezomib for 24–72 hours. Cells were collected, and total protein was extracted. IFI16 protein expression was detected by Western blotting using IFI16 primary antibody (Wuhan Sanying Biotechnology Co., Ltd.) at a concentration of 1:1000.

[0048] The results showed that bortezomib treatment dose-dependently upregulated the expression of IFI16 protein in MM cells. Figure 2 AB).

[0049] 1.3 Tissue-level detection (immunohistochemistry, IHC) Paraffin-embedded sections of bone marrow biopsies from MM patients and controls were dewaxed and antigen-retrieved, then incubated overnight at 4°C with IFI16 specific primary antibody (1:150 dilution). Detection was performed using HRP-labeled secondary antibody and the DAB chromogenic system, with hematoxylin counterstaining. The samples were observed and scored under a microscope.

[0050] The results showed that IFI16 was strongly positively stained in myeloma cells from newly diagnosed MM patients and relapsed patients, while the staining was weak or negative in control and remission samples. Figure 3 AB).

[0051] Example 2 This embodiment provides functional validation of targeting IFI16 to overcome bortezomib resistance, as detailed below: 2.1 IFI16 gene knockdown Four small interfering RNAs (siRNAs) targeting the human IFI16 gene were designed and synthesized, with the following sequences: S1: 5′-CCACCCAACAGUUCUUCAATT-3′ (SEQ ID NO: 5); S2: 5′-CAGCUUUGCCACAAACUATT-3′ (SEQ ID NO: 6); S3: 5′-GGGUAUUUAUGCUACAUAATT-3′ (SEQ ID NO:7); S4: 5′-GACCAGCCCUAUCAAGAAATT-3′ (SEQ ID NO: 8); siRNA was transfected into H929 or U266 cells using Lipofectamine 2000. Knockdown efficiency was verified by qRT-PCR and Western Blot 48-72 hours after transfection.

[0052] The results show that the designed S3 small interfering RNA sequence is a highly efficient sequence (see attached). Figure 4 (As shown in AB).

[0053] 2.2 IFI16 gene overexpression H929 or U266 cells were transduced with GFP-tagged lentiviral vectors carrying the human IFI16 gene or an empty vector control to construct a stable IFI16 overexpressing multiple myeloma cell line. After transduction, cells were cultured in medium containing 2 μg / mL puromycin for 1–2 weeks for selection.

[0054] The expression levels of IFI16 mRNA and protein were detected by qRT-PCR and Western blot to verify the overexpression efficiency.

[0055] The results showed that IFI16 expression in the lentiviral vector group was significantly higher than that in the empty vector control group (see attached). Figure 5 (As shown in AB).

[0056] 2.3 Cell proliferation and drug sensitivity assay (CCK-8 assay) H929 / U266 cells, including control (si-NC) and IFI16 knockdown (siIFI16-S3), were used at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of [number] cells / well in 96-well plates. Wells were set up as a cell control only and wells containing different concentrations of bortezomib (0, 2, 4, 8, 16, 25 nM), with three replicates per group. After 24 hours of treatment, 10 μL of CCK-8 reagent was added to each well, and after incubation for 1–2 hours, the absorbance (OD) at 450 nm was measured using a microplate reader. 450Cell viability was calculated, and dose-response curves were fitted using GraphPad Prism software to calculate the half-maximal inhibitory concentration (IC50). 50 ).

[0057] Results: IFI16 knockdown significantly inhibited the basal proliferation capacity of MM cells ( Figure 6 A), while IFI16 overexpression significantly enhanced the basal proliferative capacity of MM cells ( Figure 6 B). Meanwhile, bortezomib IC50 in siIFI16-S3 group cells... 50 The value was significantly lower than that of the si-NC group (e.g., from 16.770 nM to 7.673 nM in H929 cells), indicating that knockdown of IFI16 enhanced the sensitivity of cells to bortezomib. Figure 6 C), conversely, overexpression of IFI16 inhibited the sensitivity of cells to bortezomib ( Figure 6 D).

[0058] 2.4 Apoptosis and Cell Cycle Analysis (Flow Cytometry) Cells from the control group and the IFI16 knockdown group were treated with medium containing or without 5 nM bortezomib for 24 hours (apoptosis) or 48 hours (cell cycle). For apoptosis detection, cells were collected, double-stained with Annexin V-APC and PI, and analyzed by flow cytometry to calculate the apoptosis rate (early + late apoptosis). For cell cycle detection, cells were fixed with 70% ethanol, stained with PI, and DNA content was detected to analyze the proportions of cells in G0 / G1, S, and G2 / M phases.

[0059] Results: IFI16 knockdown itself induced apoptosis and G0 / G1 phase arrest. When combined with bortezomib, the apoptosis rate and the proportion of cells in the G0 / G1 phase were significantly higher than in the control group treated with bortezomib alone. Figure 7 A, Figure 8 A), conversely, overexpression of IFI16 inhibits apoptosis and promotes cell cycle progression (A), Figure 7 B. Figure 8 B).

[0060] Example 3 This embodiment further explores the mechanism and intervention strategies of IFI16-driven drug resistance, as detailed below: 3.1 Detection of Wnt / β-catenin pathway activity (dual-luciferase reporter gene assay) The TOPflash reporter plasmid (or the mutant control FOPflash plasmid) containing the TCF / LEF binding site was co-transfected with the internal control Renilla luciferase plasmid into control, IFI16 knockdown, or overexpressing MM cells. After 24 hours, the cells were treated with culture medium containing or without 5 nM bortezomib. After another 24 hours of culture, the cells were lysed, and the activities of firefly and Renilla luciferase were measured using a dual-luciferase reporter gene assay kit. The TOPflash / FOPflash luciferase activity ratio reflected the activity of the Wnt / β-catenin pathway.

[0061] Results: IFI16 overexpression significantly enhanced TOPflash activity, while IFI16 knockdown inhibited this activity. Bortezomib treatment further amplified this difference. Figure 9 AB).

[0062] 3.2 Validation of expression of key pathway proteins (Western Blot) MM cells under different treatments were collected, and total protein was extracted. Key proteins of the Wnt pathway were detected using specific antibodies: Wnt1, total GSK-3β, phosphorylated GSK-3β (p-GSK-3β, Ser9), β-catenin, and downstream effector molecules Cyclin D1 and p21. β-actin was used as an internal control.

[0063] Results: IFI16 overexpression upregulated the protein levels of Wnt1, p-GSK-3β, β-catenin, and Cyclin D1, and downregulated p21; IFI16 knockdown showed the opposite trend. Figure 10 AB).

[0064] 3.3 Pharmacological reversal of drug resistance In H929 cells overexpressing IFI16, pretreatment with the Wnt pathway inhibitor IWP-2 (final concentration 5 μM) for 2 hours was followed by co-treatment with different concentrations of bortezomib for 24-48 hours.

[0065] Results: IWP-2 treatment effectively reduced β-catenin levels in IFI16-overexpressing cells. Figure 11 A). The CCK-8 assay showed that IWP-2 significantly reduced the proliferation of IFI16-overexpressing cells ( Figure 11 Flow cytometry confirmed that the combination of IWP-2 and bortezomib synergistically induced apoptosis and G0 / G1 phase arrest in IFI16-overexpressing cells. Figure 12 A, B).

[0066] Example 4 This embodiment provides verification of the in vivo antitumor effect of IFI16 treatment, as detailed below: 4.1 Bloodstream dissemination model 6-8 week old NCG-immune deficient mice were randomly divided into 4 groups (n=4): 1) Vector + PBS; 2) vector + BTZ; 3) IFI16-OE + PBS; 4) IFI16-OE+BTZ.

[0067] NCG-immunodeficient mice were injected via tail vein with 2 × 10⁻⁶ luciferase-labeled U266 cells stably expressing either the empty vector or IFI16 overexpression. 6 (Number of animals / animals). Bortezomib was administered intraperitoneally at a dose of 0.5 mg / kg every 4 days; the control group received an equal volume of PBS. Systemic tumor burden was monitored periodically by in vivo bioluminescence imaging (IVIS).

[0068] Results: The tumor signal intensity in the IFI16 overexpression group was significantly higher than that in the control group, and the mice showed a poorer response to bortezomib treatment, indicating that IFI16 overexpression promotes tumor progression and leads to bortezomib resistance in vivo. Figure 13 B, C).

[0069] 4.2 Subcutaneous xenograft model Female BALB / c nude mice aged 4-6 weeks were randomly divided into 4 groups (n=4): 1) si-NC + PBS; 2) Si-NC + BTZ; 3) siIFI16-S3 + PBS; 4) siIFI16-S3 + BTZ.

[0070] H929 cells (8 × 10⁸) of control or IFI16 knockdown were subcutaneously injected into the right axilla of mice. 6 (Each tumor is approximately 100mm in size). 3 Dosing was initiated at the designated time. The bortezomib group received an intraperitoneal injection of 0.5 mg / kg every 4 days; the control group received an equal volume of PBS. The long diameter (L) and short diameter (W) of the tumor were measured periodically using calipers, calculated using the formula V = (L × W) 2 Calculate the volume by dividing the result by 2. At the end of the experiment, sacrifice the mice and weigh the tumors.

[0071] Results: Compared with the si-NC+PBS group, tumor growth was slowed in the siIFI16-S3+PBS group. The siIFI16-S3+BTZ group showed the strongest tumor growth inhibition effect, with significantly smaller terminal tumor volume and weight compared to other groups. P<0.001) Figure 13 DF). IHC of tumor tissue showed that the siIFI16-S3+BTZ group had the weakest positive staining for IFI16, Ki67, β-catenin, and Cyclin D1. Figure 13 G).

[0072] Example 5 This embodiment provides a pharmaceutical composition for treating bortezomib-resistant multiple myeloma based on the IFI16 target, and a pharmaceutical composition for overcoming IFI16-mediated bortezomib resistance based on a downstream pathway inhibition treatment strategy, as detailed below: 5.1 A pharmaceutical composition for treating bortezomib-resistant multiple myeloma. The composition comprises: Active ingredient A: siRNA targeting the human IFI16 gene (preferred sequence S3: 5′-GGGUAUUUAUGCUACAUAATT-3′), or a pharmaceutically acceptable derivative thereof, in a dose range of 0.1-100 mg / kg body weight.

[0073] Active ingredient B: bortezomib, or other pharmaceutically acceptable proteasome inhibitors, in a dose range of 0.5–2.0 mg / kg body weight.

[0074] Pharmaceutically acceptable carriers include, but are not limited to, phospholipids, cholesterol, PEG lipids used to prepare siRNA lipid nanoparticles, as well as physiological saline and glucose solutions used for injection.

[0075] This composition can be administered sequentially after separate formulation, or simultaneously encapsulated in the same delivery system (such as liposomes). Routes of administration include intravenous injection, subcutaneous injection, or local intratumoral injection.

[0076] 5.2 A pharmaceutical composition for overcoming IFI16-mediated bortezomib resistance. The composition comprises: Active ingredient A: IWP-2, an inhibitor of the Wnt / β-catenin signaling pathway, with a dosage range of 1-50 mg / kg body weight.

[0077] Active ingredient B: Bortezomib, dosage range 0.5-2.0 mg / kg body weight.

[0078] Pharmaceutically acceptable carriers include surfactants for solubilization (such as Cremophor EL), cyclodextrins, and solvents for injection.

[0079] This composition can be administered orally or by injection. The two active ingredients can be formulated as a compound preparation or administered separately for combined use during the treatment period.

[0080] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0081] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of the IFI16 gene or its expression product as a biomarker in the preparation of products for the diagnosis or prognostic assessment of multiple myeloma.

2. The application according to claim 1, characterized in that, The product is used to detect the expression level of IFI16 in samples.

3. The application according to claim 2, characterized in that, The samples included bone marrow fluid, peripheral blood, and isolated myeloma cells.

4. The application of the IFI16 gene or its expression product as a biomarker in screening or preparing products for the treatment of multiple myeloma.

5. The application according to claim 4, characterized in that, The product can overcome or reverse bortezomib resistance.

6. A pharmaceutical composition, characterized in that, The composition includes active ingredient A and active ingredient B; The active ingredient A is an IFI16 inhibitor and / or a Wnt / β-catenin signaling pathway inhibitor; The active ingredient B is bortezomib and / or other pharmaceutically acceptable proteasome inhibitors.

7. The drug according to claim 6, characterized in that, The IFI16 inhibitors include small molecule compounds, small interfering RNA, short hairpin RNA, antisense oligonucleotides, and specific antibodies targeting IFI16.

8. The drug according to claim 6, characterized in that, The Wnt / β-catenin signaling pathway inhibitor is IWP-2 or a pharmaceutically acceptable derivative thereof.

9. The drug according to claim 6, characterized in that, The drug also includes a pharmaceutically acceptable carrier; The carrier includes phospholipids, cholesterol, PEG lipids for preparing siRNA lipid nanoparticles, physiological saline and glucose solution for injection, surfactants, and cyclodextrin.

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