Stk10 diagnostic kit, stk10 inhibitor, and plk1 inhibitor for treating leukemia

Abstract

The present application relates to a kind of STK10 diagnostic kit, the application of STK10 inhibitor and PLK1 inhibitor in the treatment of leukemia.The present application finds that STK10 mRNA is significantly highly expressed in AML leukemia sample, and is positively correlated with the poor prognosis of AML patient.When STK10 gene is knocked out or inhibitor interferes with the function of STK10, it can significantly cause cell apoptosis in vitro, reduce tumor load in vivo, and prolong survival period.The curative effect is closely related to the expression amount and location of STK10 in cell, so targeted treatment can be realized according to the expression amount and location of STK10.STK10 blocks the operation of cell cycle in a synergistic or antagonistic manner during the combined treatment of SB633825 and Volasertib, leading to cell cycle disorder, thereby synergistically promoting cell apoptosis.Based on MLL-AF9 AML, MV411-GLCDX, THP1-GLCDX, ALLPDX and DLBCLPDX mouse models, the combination of the two drugs effectively prolongs the survival period of mice and reduces tumor load.The above results all show that STK10 inhibitor and PLK1 inhibitor can be used for synergistic treatment of AML and other leukemias, and have good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of STK10 diagnostic kits, STK10 inhibitors, and PLK1 inhibitors in the treatment of leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is one of the most common types of leukemia, characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. Most cases are acute and severe, with a poor prognosis; without timely treatment, it can often be life-threatening. This disease accounts for 30% of childhood leukemia. In terms of molecular biological changes and chemotherapy response, childhood AML is similar to that in adults (<50 years old). Infants and young children are more prone to developing extramedullary leukemia than adults with AML.

[0003] STK10 kinase is a unique member of the STE20 family, primarily expressed in lymphoid organs such as the spleen, thymus, and bone marrow. When co-expressed with JNK or p38 MAPK in COS7 cells, STK10 fails to activate any of these stress-activated MAPKs. Systemic STK10 knockout mice do not exhibit significant developmental defects, and their T and B lymphocytes develop normally; however, LFA-1 / ICAMs-mediated T cell aggregation is enhanced. This study suggests that STK10 regulates adhesion signaling by controlling the cell surface distribution of active LFA-1 through cytoskeleton remodeling. Furthermore, apical STK10 can phosphorylate ERM proteins, controlling the polarity of giant microvilli epithelial cells. This indicates that STK10 also plays a crucial role in maintaining epithelial integrity. Similarly, STK10 also phosphorylates ERM in lymphocytes. In STK10-deficient lymphocytes, lymphocyte migration and polarization towards chemokines are enhanced. STK10 activates PLK1 in COS-7 cells, and overexpression of STK10, a kinase-degrading kinase, impairs cell growth in 3T3 fibroblasts. Knockout of the STK10 gene in cervical cancer cell lines HeLa and Caski enhances cell migration and invasion but has no effect on ERM phosphorylation. The role of STK10 in cell death remains controversial. In Ewing sarcoma cell lines, inactivation point mutations lead to reduced apoptosis, while siRNA-mediated STK10 knockdown increases apoptosis. In conclusion, the role of STK10 kinase in the development and progression of different tumors varies, requiring further in-depth research in specific cell types and tumor environments to determine its specific function.

[0004] PLK1 is a serine / threonine protein kinase widely distributed in eukaryotic cells, participating in the initiation, maintenance, and termination of mitosis. The human PLK family includes PLK1, PLK2, PLK3, and PLK4, with PLK1 being the most extensively studied. Numerous studies have shown that PLK1 is highly expressed in various cancers, essential for rapid tumor proliferation, and associated with poor prognosis in many cancers. Therefore, PLK1 is considered an oncogene that accelerates tumor development and progression. To explore the potential of PLK1 in cancer treatment, various methods of interference have been developed, such as the use of widely available small molecule inhibitors, dominant-negative forms of PLK1, antisense oligonucleotides, and small interfering RNA.

[0005] Chinese patent CN113648425A, published on November 16, 2021, discloses a synergistic inhibitory effect of a PLK1 inhibitor and a CSNK1D / E inhibitor on tumor cells. This application demonstrates that the combination of PLK1 and CSNK1D / E kinase inhibitors can significantly inhibit the growth of liver cancer cell lines. A binary composite inhibitor can synergistically inhibit cell growth, providing a potential combination drug target for liver cancer treatment. Another Chinese patent CN102151272A, published on August 17, 2011, discloses a PLK1 inhibitor and its uses. Diseases associated with Polo-like kinase 1 (PLK1) inhibitors include melanoma, liver cancer, kidney cancer, acute leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, colorectal cancer, pancreatic cancer, ovarian cancer, breast cancer, myelodysplastic syndrome, esophageal cancer, gastrointestinal cancer, or mesothelioma.

[0006] Currently, there are no reports on the application of STK10 diagnostic kits, STK10 inhibitors, and PLK1 inhibitors in the treatment of leukemia. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing an STK10 diagnostic kit, an STK10 inhibitor, and the application of PLK1 inhibitors in the treatment of leukemia.

[0008] The first aspect provides the application of the STK10 gene or protein in the preparation of a leukemia diagnostic kit, wherein the leukemia is acute myeloid leukemia.

[0009] Secondly, a leukemia diagnostic kit is provided, the reagents of which include reagents for detecting the expression level of the STK10 gene or protein.

[0010] As a preferred example, the STK10 gene or protein is highly expressed in patients with acute myeloid leukemia.

[0011] Thirdly, it provides the application of reagents for detecting the expression level of the STK10 gene or protein in the preparation of leukemia diagnostic kits.

[0012] Fourthly, a drug for treating leukemia is provided, said drug comprising an STK10 inhibitor.

[0013] Fifthly, the application of the above-mentioned drugs in the preparation of drugs for treating leukemia, wherein the leukemia is acute myeloid leukemia.

[0014] In a sixth aspect, a pharmaceutical composition is provided, the pharmaceutical composition comprising an STK10 inhibitor and a PLK1 inhibitor.

[0015] As a preferred example, the STK10 inhibitor includes SB633825 and SLK / STK10-IN-1, and the PLK1 inhibitor includes Volasertib, Ro3280, and Rigosertib.

[0016] More preferably, the pharmaceutical composition is SB633825 and Volasetib.

[0017] In a seventh aspect, the above-mentioned pharmaceutical composition is provided for use in the preparation of a treatment for leukemia, wherein the leukemia is acute myeloid leukemia.

[0018] The advantages of this invention are:

[0019] This study confirmed that the STK10 gene can serve as a diagnostic biomarker for AML leukemia, and also demonstrated that the combination of STK10 inhibitors and PLK1 inhibitors can be used to treat leukemia, showing promising application prospects. Attached Figure Description

[0020] Appendix Figure 1 STK10 is a specific risk gene and a highly expressed gene in the pathogenesis of AML. (A, B) The intersection of three high-risk gene sets and the upregulated differential gene set (A) yields 13 genes (B); (C) A heatmap shows the HR of the 13 genes in 33 tumors, with red boxes representing pValue ≤ 0.05; (D) mRNA expression levels of each gene in TCGA AML and GTEx control samples; (E) STK10 mRNA expression levels in each tumor and corresponding normal tissue in the TCGA database; (F) STK10 mRNA expression levels in each tumor cell line in the CCLE database; (G) STK10 protein expression levels in AML patient and normal human samples; (H) STK10 protein expression levels in each tumor cell line.

[0021] Appendix Figure 2High STK10 expression is associated with poor prognosis in AML. (A) STK10 mRNA expression levels in three risk groups in the GSE14468, BEAT, and TARGET databases; (B) HR and p-values ​​of each risk factor in GSE22778; (C) Overall survival statistics of STK10 high expression and low expression groups in TCGA.

[0022] Appendix Figure 3 STK10 deficiency led to increased apoptosis in AML cells and prolonged survival in AML CDX mice. (A, B, C) MV411 cells were infected with lentiviruses containing control sgRNA and different STK10 sgRNAs. Protein expression levels in monoclonal cells were detected by Western blot (A), and apoptosis (B) and proliferation (C) were detected by flow cytometry; (D, E) 2×10 6 MV411-sgNT (n=14), MV411-sgSTK10#1 (n=13), and MV411-sgSTK10#2 (n=13) were injected into NOD-SCID mice via tail vein. In vivo imaging was used to monitor disease progression (D), and survival time was recorded (E).

[0023] Appendix Figure 4 To investigate the effects of STK10 inhibitors on increased apoptosis in AML cells and prolonged survival in AML CDX mice. (A) MV411 or MOLM13 cells were treated with SB633825 (STK10 inhibitor, 20 μM) or SLK / STK10-IN-1 (STK10 inhibitor, 20 μM), and apoptosis was measured. (B) MV411 or MOLM13 cells were treated with different concentrations of SB633825 for 48 h, and the expression of apoptosis marker proteins was detected. (C) STK10 mRNA expression in various leukemia cell lines from the CCLE database. (D, E) 5 × 10⁻⁶ mRNA was used to detect STK10 mRNA expression in AML cells. 6 MV411 was administered via tail vein injection to NOD-SCID mice (n=5), followed by intraperitoneal administration of SB633825 (40 mg / kg) or SLK / STK10-IN-1 (50 mg / kg). In vivo imaging was used to monitor disease progression (D), and survival time was recorded (E).

[0024] Appendix Figure 5The synergistic effect of SB633825 and Volasetib remained good even at low doses. (AD) MOLM13 (A) or MV411 (B) cells were treated with different low concentrations of Volasetib at nanomolar doses for 72 h, and cell viability was measured by CCK8 assay. The drug combination index (C,D) was calculated using Compusyn. CI < 0.5 indicates synergistic effect, CI < 0.3 indicates strong synergistic effect, and CI < 0.1 indicates very strong synergistic effect; (E,F) Cell lines were treated with low concentrations of SB633825 and Volasetib alone or in combination for 48 h, and cell apoptosis was measured by flow cytometry.

[0025] Appendix Figure 6 To investigate the regulation of cell cycle by STK10 and PLK1 inhibitors. (A) Cell cycle was measured after treating MV411 or MOLM13 cells with 20 μMSB633825 for different durations; (B) Cell cycle was measured after treating MV411 or MOLM13 cells with 20 μMSLK / STK10-IN-1 for different durations; (C) Cell cycle assignment diagrams for each cell population; (D) Apoptosis and cell cycle distribution were measured by flow cytometry after treating sensitive MV411 cells and insensitive THP148 h with SB633825 and Volasertib alone or in combination; (E) Cell cycle distribution was measured after treating MV411 cells with SLK / STK10-IN-1 alone or in combination.

[0026] Appendix Figure 7 The localization of STK10 differs between sensitive and insensitive strains of SB633825. (A,B) Localization of STK10 in sensitive strain (A) and insensitive strain (B) of SB633825; (C,D) Localization of PLK1 in sensitive strain (C) and insensitive strain (D) of SB633825.

[0027] Appendix Figure 8 The combination of inhibitors showed good synergistic effects in both AML CDX mouse models formed by SB633825 sensitive and non-sensitive strains. (A) Schematic diagram of mouse administration; (B,C) In vivo imaging of MV411-CDX (B) or THP1-CDX (C) mice under various administration conditions; (D,E) Statistics on the overall survival of MV411-CDX (D) or THP1-CDX (E) mice under various administration conditions.

[0028] Appendix Figure 9To demonstrate that the combination of inhibitors can delay the disease progression of MLL-AF9 AML and MLL-fusionALL PDX. (A, B) Survival statistics of MLL-AF9 AML mice under the administration mode (A); (C) Administration mode diagram of MLL-fusionALL PDX mice; (D) Monitoring of disease progression of each group of MLL-fusionALL PDX mice by in vivo imaging; (E) Statistical analysis of in vivo luminescence values ​​of each group of mice at 28 and 35 days; (F) Survival statistics of each group of MLL-fusionALL PDX mice.

[0029] Appendix Figure 10 To investigate the effect of combination inhibitors on delaying the progression of DLBCLPDX. (A) Single-drug or combination administration mode; (B) 20 days after administration, mice were euthanized, tumors were harvested and the volume of each group was counted; (C) Changes in tumor volume in each group throughout the disease progression; (D) Display of tumor masses in each group. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0031] In the following embodiments, unless otherwise specified, the instruments, reagents, materials, etc. involved are all conventional instruments, reagents, materials, etc. in the prior art, which can be obtained through regular commercial channels. Unless otherwise specified, the experimental methods and detection methods involved are all conventional experimental methods and detection methods that already exist in the prior art.

[0032] Example 1

[0033] 1. Experimental Materials

[0034] 1.1 Cell sources: MV411, THP1 (Shanghai Institute of Life Sciences, Chinese Academy of Sciences Cell Bank); REH, SEM (ATCC); MOLM13 (DSMZ).

[0035] 1.2 Small molecule compounds: SB633825 (HY-108333, MCE); SLK / STK10-IN-1 (HY-132868, MCE); Busulfan (NSC-750) (S1692, Selleck); Volasertib (BI 6727) (S2235, Selleck); Rigosertib (ON-01910) (S1362, Selleck); Ro3280 (S7248, Selleck).

[0036] 1.3 Plasmid vectors: pSPAX2, pMD2G (Addgene12260), lentiCRISPRv2 (Shanghai Xinzhuo Biotechnology Co., Ltd.), CRISPRv2-hSTK101 / 2 / 3 / 4, pCDH-Luciferase-T2A-GFP.

[0037] 1.4 Primer Sequences:

[0038] All primers were synthesized at Shanghai Sangon Biotech Co., Ltd., and their specific names and sequences are as follows:

[0039]

[0040] 1.5 Construction of animal models

[0041] MLL-AF9 mouse model: MLL-AF9 cDNA was cloned into MigR1-GFP to obtain the MigR1-GFP-MLL-AF9 plasmid, which was then transfected into 293T cells and packaged into a retrovirus. C57BL / 6 mice were euthanized by cervical dislocation, and bone marrow cells were obtained. Lineage-(Ter119-,CD3-,B220-,CD11b-,Gr-1-) bone marrow cells were sorted and then resuspended in IMDM complete medium (10% FBS, 50 ng / mL mSCF, 10 ng / mL mIL-6, 6 ng / mL mIL-3, 4 μg / mL polybrene). The cells were then infected with the above retrovirus for 48 h. GFP cells were sorted. + A group of mice were injected via tail vein into C57BL / 6 mice that had been previously treated with busulfan (20 mg / kg). Four to five weeks later, the mice were sacrificed when near death, and bone marrow cells were collected for GFP sorting. + MLL-AF9 cells were then inoculated into untreated C57BL / 6 mice to establish an MLL-AF9 mouse model.

[0042] CDX Mouse Model: Taking the construction of the MV411-sgSTK10 CDX mouse model as an example, this paper illustrates the construction method of the CDX mouse model. The pCDH-Luciferase-T2A-GFP plasmid was packaged into GL virus, and the MV411-sgSTK10 monoclonal cell line was infected with GL virus. After 48 hours of infection, GFP+MV411-sgSTK10 cells were sorted by flow cytometry and then expanded. 2×10⁶ cells were then cultured... 6 GFP + MV411-sgSTK10 cells were transfused via tail vein into NOD-SCID mice that had been treated with busulfan (20 mg / kg) one day prior. In vivo imaging was used to detect GFP levels in the mice 10 days later. + Infiltration status of MV411-sgSTK10 cells.

[0043] PDX mouse model: After preparing busulfan injection, mice were intraperitoneally injected with busulfan (100 μL / mouse) for myeloablation. Frozen leukemia patient samples were resuscitated, stained with anti-CD3 antibody and Zombie stain, and CD3-Zombie- cells were sorted by flow cytometry. Cells were resuspended in IMDM complete medium (IMDM + 50 ng / mL SCF + 6 ng / mL IL-3 + 10 ng / mL IL-6 + 10% FBS + 1% P / S) and seeded in 6-well plates. On Day 1, cells from the 6-well plates were collected, washed twice with pre-chilled PBS, and then injected via tail vein into mice at a dose of 2 × 10⁻⁶ cells per mouse. 6 On Day 40, the number of leukemia blast cells in peripheral blood was monitored. Blood was collected from the tail vein of mice, treated with erythrocyte lysis buffer, and then stained by flow cytometry with anti-human CD45 and anti-mouse CD45 to count the human CD45 content. + Cell ratio;

[0044] 2. Statistical Analysis

[0045] Experimental data are presented as mean ± standard error. All data were statistically analyzed using GraphPad Prism 6.0 software. Unpaired Student's t-tests were used to compare two groups. For larger-than-life data sets where each data point is a single-factor variable, one-way ANOVA was used for significance analysis. When multiple groups involved multiple variables, two-way ANOVA was used to calculate the corresponding p-values. *P < 0.05 indicates a significant difference between groups, and **P < 0.01 indicates a highly significant difference between groups.

[0046] 3 Experimental Results

[0047] 3.1 STK10 as an AML-specific risk gene and a highly expressed gene

[0048] We analyzed the risk (Hazard Ratio, HR) genes (i.e., HR≥1 and pValue≤0.05) of TCGAAML, TARGETAML, and OHSUAML, as well as the differentially expressed genes between TCGAAML and GTEx, and took the intersection of the four datasets. Figure 1 A), a total of 13 genes were obtained ( Figure 1 B). A review of the overall expression of these 13 genes in pan-cancer studies revealed that they are all high-risk genes for AML, and the differences were statistically significant. Figure 1 C). Compared with normal GTEx samples, all 13 genes were significantly overexpressed in TCGA AML, with STK10 showing the highest expression level and the largest fold change. Figure 1 D). STK10 was expressed at the highest level in AML compared to other tumor samples. Figure 1 E). Compared with other tumor cell lines, STK10 expression was also highest in AML cell lines. Figure 1 F). Western blot results also showed that STK10 protein expression levels were significantly higher in AML samples compared to healthy samples. Figure 1 G); compared with other tumor cell lines, the protein expression level of STK10 in AML cell lines is also significantly higher (G); Figure 1 The above results all indicate that STK10 is an AML-specific risk gene and a highly expressed gene.

[0049] 3.2 High STK10 expression is associated with poor prognosis in AML

[0050] The GSE14468, BEAT, and TARGETAML databases all show that higher STK10 expression levels correlate with worse prognosis. Figure 2 A). Similarly, the GSE22778 dataset also shows that high expression of STK10 and PLK1 is a significant risk factor. Figure 2 B). When TCGAAML samples were divided into two groups, one with high STK10 expression and the other with low STK10 expression, the overall survival of the samples in the high STK10 expression group was significantly reduced (B). Figure 2 C). In general, high STK10 expression is associated with poor prognosis in AML.

[0051] 3.3 STK10 is a key factor for AML cell survival.

[0052] When the STK10 gene in MV411 is knocked out ( Figure 3A) Apoptosis was significantly increased in the STK10 gene knockout monoclonal cell lines MV411-sgSTK10#1 and MV411-sgSTK10#2. Figure 3 B), but proliferation did not change significantly. Figure 3 C). When CDX mouse models were constructed using MV411-sgSTK10#1 and MV411-sgSTK10#2, the tumor burden in the knockout mice was significantly reduced. Figure 3 D), survival time is significantly prolonged ( Figure 3 E). Therefore, STK10 is a key factor for AML cell survival.

[0053] 3.4 STK10 inhibitors induce apoptosis in AML cells and prolong the survival of AML CDX mice.

[0054] When MV411 or MOLM13 was treated with SB633825 or SLK / STK10-IN-1, the apoptosis rate in the drug-treated groups was significantly higher than that in the control group. Figure 4 A). When treated with different concentrations of SB633825, the expression of apoptosis marker proteins was upregulated. Figure 4 B). CCLE data showed that the expression levels of other targets of SB633825 (TIE2 and BRK) were extremely low in various leukemia cell lines. Figure 4 C), which means that SB633825 primarily functions by targeting STK10 in leukemia cells. An AML mouse model was established by injecting NOD-SCID immunodeficient mice with MV411. When SB633825 and SLK / STK10-IN-1 were administered, respectively, the tumor cell load in the inhibitor group was significantly reduced compared to the control group. Figure 4 D), survival time is significantly prolonged ( Figure 4 E). These results all indicate that STK10 has an anti-apoptotic effect in AML cell lines and is crucial for maintaining the survival of AML cells.

[0055] 3.5 STK10 inhibitors and PLK1 inhibitors have synergistic effects.

[0056] Further reductions in the doses of SB633825 and Volasetib, even down to nanomolar levels of SB633825, were observed. When the Volasetib dose was ≤10 nM, while MOLM13 showed no response with increasing SB633825 dose, MV411 did exhibit some response at SB633825 concentrations up to 1 μM. When the Volasetib dose was greater than 10 nM, both MOLM13 and MV411 showed dose-dependent responses with increasing SB633825 dose. Figure 5 A, 5B).

[0057] According to the CI values, 20 nM Volasertib and 1 μM SB633825 showed a good synergistic effect in MV411 cells; in MOLM13 cells, 40 nM Volasertib and 800 nM SB633825 also showed a good synergistic effect. Figure 5 C,5D). When THP1 and MV411 are treated with low doses of inhibitors in combination, they synergistically promote apoptosis. Figure 5 E), similar results were also found in the ALL cell line REH and SEM. Figure 5 F). Therefore, STK10 inhibitors and PLK1 inhibitors can significantly synergistically promote apoptosis.

[0058] 3.6 STK10 inhibitors and PLK1 inhibitors regulate cell cycle progression

[0059] When MV411 and MOLM13 were treated with SB633825, respectively, the cell cycle was arrested in the G0 / G1 phase. Figure 6 A). When MV411 and MOLM13 were treated with SLK / STK10-IN-1, respectively, the cell cycle was arrested in the G2 / M phase in the early stage and in the G0 / G1 phase in the late stage. Figure 6 B). In MV411, SB633825 and Volasertib mutually deactivate cell cycle arrest; while in THP1, SB633825 and Volasertib synergistically arrest the cell cycle at the G2 / M phase. Figure 6 (D,6E). Although the regulatory mechanisms of the cell cycle differ in different AML cell lines, the combined use of inhibitors ultimately leads to cell cycle disorder and ultimately apoptosis.

[0060] 3.7 STK10 localization differs in cell lines with varying sensitivities to combination inhibitors.

[0061] Cells can be classified into three categories based on their sensitivity to the combination of inhibitors: ① Single-drug sensitive, combination-more sensitive: These cells are sensitive to single-drug treatment, inducing significant apoptosis and cell cycle arrest. Combination therapy further enhances their sensitivity, leading to even more pronounced apoptosis and cell cycle arrest, such as MV411, NOMO1, MOLM13, U937, and SEM; ② Single-drug insensitive, combination-sensitive: These cells are insensitive to single-drug treatments and do not induce significant apoptosis or cell cycle arrest, but are highly sensitive to combination therapy, resulting in significant apoptosis and cell cycle arrest, such as THP1 and Raji; ③ Single-drug and combination-insensitive: These cells are insensitive to both single-drug and combination therapy, failing to induce significant apoptosis and cell cycle arrest, such as SKM1. Most AML cells belong to category ①, showing high sensitivity to combination therapy; a small portion of AML cells belong to category ②, showing high sensitivity to combination therapy even if insensitive to single-drug therapy; only a very small number belong to category ③, showing no response to combination therapy. To further investigate this mechanism, the localization of STK10 and PLK1 was detected by immunofluorescence. The results showed that STK10 was significantly more abundant in the cell membrane and cytoplasm than in the nucleus in type ① cells. Figure 7 A), while the distribution of STK10 in cells of type ② and type ③ is exactly the opposite ( Figure 7 B), which suggests that cellular sensitivity to inhibitors may be related to the localization pattern of STK10. Meanwhile, the distribution of PLK1 was not significantly different in cell types ①, ②, and ③. Figure 7 There was no significant difference in expression levels (C, 7D). Therefore, the sensitivity of the combination therapy is mainly determined by the localization of STK10. In inhibitor-sensitive cells, the expression level of STK10 also affects the cell's sensitivity. Figure 1 Therefore, targeted therapy can be achieved based on the expression level and location of STK10.

[0062] 3.8 Low-dose dual-drug combination therapy significantly prolonged the survival of CDX mice

[0063] A CDX mouse model was established by intravenously injecting NOD-SCID immunodeficient mice with MV411-GL (GFP-Luciferase) and THP1-GL. The specific administration route is as follows: Figure 8 A. In vivo imaging was used to monitor tumor load in mice. The imaging results showed that, compared to the control group, the combination therapy group had a significantly lower tumor load and a significantly longer survival time. Figure 8 B, 8C). The survival time of THP1-GL mice was extended by approximately 15 days, and that of MV411-GL mice was extended by approximately 20 days. Figure 8 (D,8E). Therefore, low-dose dual-drug combination therapy can significantly prolong the survival of CDX mice.

[0064] 3.9 Low-dose dual-drug combination therapy showed good efficacy in mouse AML and ALL PDX models.

[0065] Lineage-(Ter119-,CD3-,B220-,CD11b-,Gr-1-) bone marrow cells were infected with mouse MLL-AF9 retrovirus, and GFP was sorted. + A population was established by injecting the drug via tail vein into C57BL / 6 mice to create an MLL-AF9 mouse leukemia model. In this model, the drug was administered as a single agent or in combination (…). Figure 9 A) It was found that Volasetib monotherapy effectively prolonged the survival of mice, while combination therapy further prolonged the overall survival of mice. Figure 9 B).

[0066] In addition, ALL PDX mice were treated with single or combination therapy. Figure 9 C). The results showed that, compared with the control group, the SB633825 group and the Volasertib group had poor monotherapy effects due to dosage issues, and there was no significant difference in tumor burden. However, the dual-drug group showed good efficacy, and the tumor burden was significantly reduced throughout the course of leukemia. Figure 9 D). On days 28 and 35 after administration, the fluorescence values ​​in the combination group also decreased significantly ( Figure 9 E), the overall survival of mice was significantly prolonged ( Figure 9 F). These results all indicate that low-dose inhibitor combination therapy still has good efficacy in the ALL PDX model.

[0067] 3.10 Low-dose dual-drug combination therapy showed promising efficacy in a mouse DLBCL PDX model.

[0068] We constructed a DLBCL PDX model in NOD-SCID mice by subcutaneous tumor-bearing DLBCL patient samples, and administered the drug as a single agent or in combination. Figure 10 A). At low doses, SB633825 monotherapy inhibited tumor growth, while the Volasertib group showed some tumor regression. The regression was more pronounced in the combination therapy group, with tumors in several mice shrinking from 1 cm at the time of treatment. 2 Size completely disappeared ( Figure 10 (BD). This demonstrates the remarkable potential of combining STK10 with PLK1 inhibitors in the treatment of solid tumors.

[0069] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition is SB633825 and Volasetib.

2. The use of the pharmaceutical composition according to claim 1 in the preparation of a treatment for leukemia, characterized in that, The leukemia mentioned is acute myeloid leukemia.

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